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NPTEL – Biotechnology – Microbiology Module 6 – Microbial Metabolism Lecture 1 – Overview of microbial metabolism Metabolism refers to the sum of all chemical reactions within a living organism. Chemical reactions either release or require energy. Metabolism can be viewed as an energy-balancing act. Metabolism Release energy (catabolism) Require energy (anabolism) Catabolism– enzyme-regulated chemical reactions that release energy. Complex organic compounds are broken down into simpler ones. These reactions are called catabolic or degradative reactions. They are generally hydrolytic reactions (reactions that use water and in which chemical bonds are broken), and they are exergonic (produce more energy than they consume). Ex. Cells break down sugars into CO2and H2O. Anabolism – enzyme-regulated energy requiring reactions. The building of complex organic molecules from simpler ones. These reactions are called anabolic or biosynthetic and they are generally dehydration synthesis reactions (reactions that release water), and they are endergonic (consume more energy than they produce). Ex. Formation of proteins from amino acids, nucleic acids from nucleotides, polysaccharides from simple sugars) These reactions generate the materials for growth. This coupling of energy requiring and energy-releasing reactions is made possible through the molecule adenosine triphosphate (ATP). ATP stores energy derived from catabolic reactions and releases it later to drive anabolic reactions and perform other cellular work. Fig. 1. ATP molecule Joint initiative of IITs and IISc – Funded by MHRD Page 1 of 73 NPTEL – Biotechnology – Microbiology ATP – an adenine, a ribose and 3 phosphate groups. When terminal phosphate group is split from ATP, ADP is formed, and energy is released to drive anabolic reactions. ATP ADP + Pi + energy Then, energy from catabolic reactions is used to combine ADP and a P to resynthesize ATP. ADP + Pi + energy ATP Anabolic reactions – coupled to ATP breakdown Catabolic reactions – ATP synthesis. ENZYMES: • Substances that can speed up a chemical reaction without being permanently altered themselves are called catalysts. • In living cells, enzymes serve as biological catalysts. • Enzymes are specific and act on specific substances called the enzyme substrate(s) and each catalyzes only one reaction. Ex. Sucrose is the substrate of the enzyme sucrose, which catalyzes the hydrolysis of sucrose to glucose and fructose. Enzyme specificity and efficiency: Enzymes are large globular proteins that range in MW from about 10,000 to several million. Each enzyme has a characteristic three-dimensional shape with a specific surface configuration as a result of its primary, secondary and tertiary structures. This enables it to find the correct substrate from among the large number of diverse molecules in the cell. Enzymes are extremely efficient. Under optimum conditions can catalyze reactions at rates 108 to 1010 times higher than those of comparable reactions without enzymes. Turnover number (maximum number of substrate molecules an enzyme molecule converts to product each second) is between 1 and 10,000 and can be high as 500,000. Joint initiative of IITs and IISc – Funded by MHRD Page 2 of 73 NPTEL – Biotechnology – Microbiology Enzyme components: • Some enzymes consist entirely of proteins. • Most consist of both a protein portion called an apoenzyme and a nonprotein component called a cofactor. • Ions of iron, zinc, magnesium or calcium are examples of cofactors. If the cofactor is an organic molecule, it is called a coenzyme. • Together, the apoenzyme and cofactor form a holoenzyme, or whole active enzyme. If the cofactor is removed, the apoenzyme will not function. • Coenzymes assist the enzyme by accepting atoms removed from the substrate or by donating atoms required by the substrate. Some act as electron carriers, removing electrons from the substrate and donating them to other molecules in subsequent reactions. • Many coenzymes are derived from vitamins. Two of the most important coenzymes in cellular metabolism are Nicotinamide adenine dinucleotide (NAD+) Nicotinamide adenine dicucleotide phosphate (NADP+) These contain derivatives of B vitamin nicotinic acid (niacin) NAD+ - involved in catabolic (energy-yielding reactions) NADP+ - involved in anabolic (energy-requiring reactions) • Flavin coenzymes, such as flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), contains derivatives of the B vitamin riboflavin and are also electron carriers. • Coenzyme A (CoA) contains derivatives of pantothenic acid, another B vitamin. This plays an important role in the synthesis and breakdown of fats and in a series of oxidizing reactions called the Krebs cycle. • Some cofactors are metal ions, including Fe, Cu, Mg, Mn, Zn, Ca and Co. They form a bridge between the enzyme and a substrate. Ex. Mg2+ is required by many phosphorylating enzymes (enzymes that transfer a phosphate group from ATP to another substrate). Joint initiative of IITs and IISc – Funded by MHRD Page 3 of 73 NPTEL – Biotechnology – Microbiology Energy production: There are two general aspects of energy production; the concept of oxidation-reduction and the mechanism of ATP generation. Oxidation – reduction reactions: Oxidation – is removal of electron from an atom or molecule, a reaction that often produces energy. Reduction – is addition of one or more electrons to an atom or molecule. Oxidation and reductions reactions are always coupled. The pairing of these reactions is called oxidation-reduction or redox reactions. Most biological oxidation reactions involve the loss of hydrogen atoms, they are also dehydrogenation. Hydrogen atom contains both electrons and protons and in cellular oxidations, electrons and protons are removed at the same time. An organic molecule is oxidized by the loss of two hydrogen atoms, and a molecule of NAD+ is reduced by accepting two electrons and one proton. One proton is left over and is released into the surrounding medium. The reduced coenzyme contains more energy than NAD+. This energy can be used to generate ATP in later reactions. Cells use oxidation- reduction (biological) reactions in catabolism to extract energy from nutrient molecules. Ex. Cell oxidizes a molecule of glucose to Co2 and H2O. The energy in the glucose molecule is removed in stepwise manner and ultimately is trapped by ATP, which can then serve as an energy source for energy requiring reactions. Thus glucose is a valuable nutrient for organisms. The generation of ATP: Much of the energy released during oxidation – reduction reactions is trapped within the cell by the formation of ATP. A phosphate group is added to ADP with the input of energy to form ATP. Addition of a phosphate to a chemical compound is called phosphorylation. Organisms use three mechanisms of phosphorylation to generate ATP from ADP. Substrate – level phosphorylation:ATP is generated when a high energy phosphate is directly transferred from a phosphorylated compound (a substrate) to ADP. Generally the Joint initiative of IITs and IISc – Funded by MHRD Page 4 of 73 NPTEL – Biotechnology – Microbiology phosphate has acquired its energy during an earlier reaction in which the substrate itself was oxidized. C-C-C~ P + ADP C-C-C + ATP Oxidative phosphorylation: Electrons are transferred from organic compounds to one group of electron carriers (usually to NAD+ and FAD). Then, the electrons are passed through a series of different electron carriers to molecules of O2 or other oxidized inorganic and organic molecules. This process occurs in the plasma membrane of prokaryotes and in the inner mitochondrial membrane of eukaryotes. The sequence of electron carriers used in oxidative phosphorylation is called an electron transport chain. The transfer of electrons from one electron carrier to the next releases energy, some of which is used to generate ATP from ADP through a process called chemiosmosis. Photophosphorylation:Occurs only in photosynthetic cells which contain light-trapping pigments such as chlorophylls. In photosynthesis, organic molecules, especially sugars, are synthesized with the energy of light from the energy-poor building blocks Co2 and H2O. Photophosphorylation starts this process by converting light energy to the chemical energy of ATP and NADPH, which in turn, are used to synthesize organic molecules. As in oxidative phosphorylation, an electron transport chain is involved. All microbial metabolisms can be arranged according to three principles: 1. How the organism obtains carbon for synthesizing cell mass? • autotrophic – carbon is obtained from carbon dioxide (CO2) • heterotrophic – carbon is obtained from organic compounds • mixotrophic – carbon is obtained from both organic compounds and by fixing carbon dioxide 2. How the organism obtains reducing equivalents used either in energy conservation or in biosynthetic reactions: • lithotrophic – reducing equivalents are obtained from inorganic compounds • organotrophic – reducing equivalents are obtained from organic compounds Joint initiative of IITs and IISc – Funded by MHRD Page 5 of 73 NPTEL – Biotechnology – Microbiology 3. How the organism obtains energy for living and growing: • chemotrophic – energy is obtained from external chemical compounds • phototrophic – energy is obtained from light. • chemolithoautotrophs obtain energy from the oxidation of inorganic compounds and carbon from the fixation of carbon dioxide. Examples: Nitrifying bacteria, Sulfur-oxidizing bacteria, Iron-oxidizing bacteria, Knallgas-bacteria • photolithoautotrophs obtain energy from light and carbon from the fixation of carbon dioxide, using reducing equivalents from inorganic compounds. Examples: Cyanobacteria (water (H2O) as reducing equivalent donor), Chlorobiaceae, Chromatiaceae (hydrogen sulfide (H2S) as reducing equivalent donor), Chloroflexus (hydrogen (H2) as reducing equivalent donor) • chemolithoheterotrophs obtain energy from the oxidation of inorganic compounds, but cannot fix carbon dioxide (CO2). Examples: some Thiobacilus, some Beggiatoa, some Nitrobacter spp., Wolinella (with H2 as reducing equivalent donor), some Knallgas-bacteria, some sulfate-reducing bacteria • chemoorganoheterotrophs obtain energy, carbon, and reducing equivalents for biosynthetic reactions from organic compounds. Examples: most bacteria, e. g. Escherichia coli, Bacillus spp., Actinobacteria • photoorganoheterotrophs obtain energy from light, carbon and reducing equivalents for biosynthetic reactions from organic compounds. Some species are strictly heterotrophic, many others can also fix carbon dioxide and are mixotrophic. Examples: Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodomicrobium, Rhodocyclus, Heliobacterium, Chloroflexus (alternatively to photolithoautotrophy with hydrogen). Diversity of electron acceptors for respiration Organic compounds: – Eg. fumarate, dimethylsulfoxide (DMSO), Trimethylamine-N-oxide (TMAO) Inorganic compounds: – Eg. NO3-, NO2-, SO42-, S0, SeO42-, AsO43- Joint initiative of IITs and IISc – Funded by MHRD Page 6 of 73 NPTEL – Biotechnology – Microbiology Metals: – Eg. Fe3+, Mn4+, Cr6+ Minerals/solids: – Eg. Fe(OH)3, MnO2 Gasses: – Eg. NO, N2O, CO2 Most microorganisms oxidize carbohydrates as their main source of cellular energy.Microorganisms use two general processes: Cellular respiration and fermentation. Microorganisms also use anaerobic pathway to oxidize glucose. In case of aerobic respiration, the ultimate e- acceptor is O2 and the reduced form is H2O.There are four stages of aerobic respiration: Oxygen extracts chemical energy from glucose, the glucose molecule must be split up into two molecules of pyruvate. This process also generates two molecules of adenosine triphosphate as an immediate energy yield and two molecules of NADH. C6H12O6 + 2 ADP + 2 Pi + 2 NAD+ → 2 CH3COCOO− + 2 ATP + 2 NADH + 2 H2O + 2H+ • The citric acid cycle begins with the transfer of a two-carbon acetyl group from acetyl-CoA to the four-carbon acceptor compound (oxaloacetate) to form a sixcarbon compound (citrate). • The citrate then goes through a series of chemical transformations, losing two carboxyl groups as CO2. The carbons lost as CO2 originate from what was oxaloacetate, not directly from acetyl-CoA. The carbons donated by acetyl-CoA become part of the oxaloacetate carbon backbone after the first turn of the citric acid cycle. Loss of the acetyl-CoA-donated carbons as CO2 requires several turns of the citric acid cycle. However, because of the role of the citric acid cycle in anabolism, they may not be lost, since many TCA cycle intermediates are also used as precursors for the biosynthesis of other molecules. • Most of the energy made available by the oxidative steps of the cycle is transferred as energy-rich electrons to NAD+, forming NADH. For each acetyl group that enters the citric acid cycle, three molecules of NADH are produced. Joint initiative of IITs and IISc – Funded by MHRD Page 7 of 73 NPTEL – Biotechnology – Microbiology • Electrons are also transferred to the electron acceptor Q, forming QH2. • At the end of each cycle, the four-carbon oxaloacetate has been regenerated, and the cycle continues. Anaerobic respiration - Microbes are capable of using all sorts of other terminal electron accepters besides oxygen. A few examples of anaerobic respiration; • Final electron acceptor is an inorganic substance other than O2. • Some bacteria such as Pseudomonas and Bacillus can use a nitrate ion (NO-3), in the presence of an enzyme called nitrate reductase, as a final electron acceptor, the nitrate ion is reduced to nitrite ion (NO2-). • Nitrite ion can be converted to nitrous oxide (N2O), or nitrogen gas (N2) (denitrification process) which helps in recycling of nitrogen. • Other bacteria like Desulfovibrio use sulfate (SO42-) as the final electron acceptor and forms hydrogen sulfide (H2S). • Still other bacteria use carbonate (CO32-) to form methane (CH4). • Anaerobic respiration by bacteria using nitrate and sulfate as final electron acceptors is essential for the nitrogen and sulfur cycles that occur in nature. • Amount of ATP generated varies with the organisms and the pathway. Because only a part of the Krebs cycle operates and since not all the carriers in the electron transport chain participate, ATP yield is less and accordingly anaerobes tend to grow more slowly than aerobes. Microbial fermentation Fermentation is a specific type of heterotrophic metabolism that uses organic carbon instead of oxygen as a terminal electron acceptor. This means that these organisms do not use an electron transport chain to oxidize NADH to NAD+ and therefore must have an alternative method of using this reducing power and maintaining a supply of NAD+ for the proper functioning of normal metabolic pathways (e.g. glycolysis). As oxygen is not required, fermentative organisms are anaerobic. Many organisms can use fermentation under anaerobic conditions and aerobic respiration when oxygen is present. These organisms are facultative anaerobes. To Joint initiative of IITs and IISc – Funded by MHRD Page 8 of 73 NPTEL – Biotechnology – Microbiology avoid the overproduction of NADH, obligately fermentative organisms usually do not have a complete citric acid cycle. Instead of using an ATP synthase as in respiration, ATP in fermentative organisms is produced by substrate-level phosphorylation where a phosphate group is transferred from a high-energy organic compound to ADP to form ATP. As a result of the need to produce high energy phosphate-containing organic compounds (generally in the form of CoA-esters) fermentative organisms use NADH and other cofactors to produce many different reduced metabolic by-products, often including hydrogen gas (H2). These reduced organic compounds are generally small organic acids and alcohols derived from pyruvate, the end product of glycolysis. Examples include ethanol, acetate, lactate, and butyrate. Fermentative organisms are very important industrially and are used to make many different types of food products. The different metabolic end products produced by each specific bacterial species are responsible for the different tastes and properties of each food. The two main types of fermentation are alcoholic fermentation and lactic acid fermentation (Fig.2). The two main types of fermentation are: 1) Alcoholic fermentation 2) Lactic acid fermentation Fig. 2. Lactic acid and ethanolic fermentations Joint initiative of IITs and IISc – Funded by MHRD Page 9 of 73 NPTEL – Biotechnology – Microbiology Both types have the same reactants: Pyruvic acid and NADH, both of which are products of glycolysis. In alcoholic fermentation, the major products are alcohol and carbon dioxide. In lactic acid fermentation, the major product is lactic acid. For both types of fermentation, there is a side product: NAD+ which is recycled back to glycolysis so that small amounts of ATP can continue to be produced in the absence of oxygen. The chemical equations below summarize the fermentation of sucrose, whose chemical formula is C12H22O11. One mole of sucrose is converted into four moles of ethanol and four moles of carbon dioxide: C12H22O11 +H2O + invertase →2 C6H12O6 C6H12O6 + Zymase → 2C2H5OH + 2CO2 The process of lactic acid fermentation using glucose is summarized below. In homolactic fermentation, one molecule of glucose is converted to two molecules of lactic acid:[3] C6H12O6 → 2 CH3CHOHCOOH In heterolactic fermentation, the reaction proceeds as follows, with one molecule of glucose converted to one molecule of lactic acid, one molecule of ethanol, and one molecule of carbon dioxide: C6H12O6 → CH3CHOHCOOH + C2H5OH + CO2 Joint initiative of IITs and IISc – Funded by MHRD Page 10 of 73 NPTEL – Biotechnology – Microbiology REFERENCES: Text Books: 1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition). Jones and Bartlett Student edition. 2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An Introduction. Benjamin Cummings. Reference Books: 1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw Hill companies. Joint initiative of IITs and IISc – Funded by MHRD Page 11 of 73 NPTEL – Biotechnology – Microbiology Module 6 – Microbial Metabolism Lecture 2 – Carbohydrate Catabolism Most microorganisms oxidize carbohydrates as their primary source of cellular energy. Glucose is the most common carbohydrate energy source used by cells. To produce energy from glucose microorganisms use two general processes: cellular respiration and fermentation. Anaerobic respiration is another mode where the final electron acceptor is an inorganic substance other than oxygen. Catabolism/Oxidation of carbohydrates or Aerobic respiration of carbohydrates: – Most efficient way to extract energy from glucose. Occurs in three principal stages: 1. Glycolysis 2. Kreb Cycle 3. Electron transport chain Glycolysis – Oxidation of glucose to pyruvic acid with the production of some ATP and energy containing NADH. Krebs cycle – Oxidation of acetyl (a derivative of pyruvic acid) to Co2, with the production of some ATP, energy containing NADH, and another reduced electron carrier, FADH2. Electron Transport chain – NADH and FADH2 are oxidized, contributing the electrons, they have carried from the substrate to a ‘cascade’ of oxidation-reduction reactions involving a series of additional electron carriers. Energy from these reactions is used to generate a considerable amount of ATP. In respiration, most of the ATP is generated in this step. Fermentation: Initial stage is also glycolysis which produces pyruvic acid. But pyruvic acid is converted into one or more different products, depending on the type of cell. These products might include alcohol and lactic acid. Unlike respiration, there is no Krebs cycle or electron transport chain. Accordingly, the ATP yield is also much lower. Joint initiative of IITs and IISc – Funded by MHRD Page 12 of 73 NPTEL – Biotechnology – Microbiology Glycolysis Or Embden-Meyerhof (EMF) pathway: In glycolysis (from the Greek glykys, meaning “sweet,”and lysis, meaning “splitting”), a molecule of glucose is degraded in a series of enzyme-catalyzed reactions to yield two molecules of the three-carbon compound pyruvate. During glycolysis NAD+ is reduced to NADH and there is a net production of 2 ATP molecules by substrate level phosphorylation. Glycolysis does not require oxygen and can occur whether present or not. Reactions in glycolytic pathway Glycolysis involves 10 enzymatic reactions, summarized in Figure 1: 1. The phosphorylation of glucose at position 6 by hexokinase, 2. The isomerization of glucose-6-phosphate to fructose-6-phosphate by phosphohexose isomerase, 3. The phosphorylation of fructose-6-phosphate to fructose-1,6bisphosphate by phosphofructokinase, 4. The cleavage of fructose-1,6-bisphosphate by aldolase. This yields two different products, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, 5. The isomerization of dihydroxyacetone phosphate to a second molecule of glyceraldehyde phosphate by triose phosphate isomerase, 6. The dehydrogenation and concomitant phosphorylation of glyceraldehyde-3-phosphate to 1,3-bis-phosphoglycerate by glyceraldehyde-3phosphate dehydrogenase, 7. The transfer of the 1-phosphate group from 1,3-bis-phosphoglycerate to ADP by phosphoglycerate kinase, which yields ATP and 3phosphoglycerate, 8. The isomerization of 3-phosphoglycerate to 2-phosphoglycerate by phosphoglycerate mutase, 9. The dehydration of 2-phosphoglycerate to phosphoenolpyruvate by enolase. 10. The transfer of the phosphate group from phosphoenolpyruvate to ADP by pyruvate kinase, to yield a second molecule of ATP. Joint initiative of IITs and IISc – Funded by MHRD Page 13 of 73 NPTEL – Biotechnology – Microbiology Fig. 3. Glycolysis. (Source, Lehninger, Principles of Biochemistry, Fifth Edition) Joint initiative of IITs and IISc – Funded by MHRD Page 14 of 73 NPTEL – Biotechnology – Microbiology Overall reaction of glycolysis Glucose +2NAD+ + 2ADP + 2Pi ----2 pyruvate + 2NADH + 2H+ +2ATP + 2H2O Because 2 moleucles of ATP were needed to get glycolysis started and four molecules of ATP are generated by the process, there is a net gain of two molecules of ATP for each molecule of glucose that is oxidised. Alternatives of Glycolysis: Many bacteria have another pathway in addition to glycolysis for the oxidation of glucose. The most common are i) pentose phosphate pathway and ii) Entner-Doudoroff pathway 1. Pentose Phosphate pathway (Hexose monophosphate shunt): This provides a means for the breakdown of five-carbon sugars (pentoses) as well as glucose. A key feature is that it produces important intermediates pentoses used in the synthesis of nucleic acids, glucose from Co2 in photosynthesis and certain amino acids. The pathway is an important producer of the reduced coenzyme NADPH from NADP+. This pathway yields a net gain of only one molecule of ATP for each molecule of glucose oxidised. Bacteria that use this pathway include Bacillus subtilis, E.coli, Leuconostoc mesenteroides and Enterococcus faecalis. The Entner-Doudoroff pathway: For each molecule of glucose this pathway produces 2 molecules of NADPH and one molecule of ATP for use in cellular biosynthetic reactions. Bacteria that have the enzymes for this pathway can metabolize glucose without either glcolysis or the pentose phosphate pathway. Found in some gram-negative bacteria, including Rhizobium, Pseudomonas and Agrobacterium; generally not found among gram-positive bacteria. Cellular/Aerobic respiration After glucose has been broken down to pyruvic acid, the pyruvic acid can be channeled into the next step of either fermentation or cellular respiration. Cellular respiration – is defined as an ATP generating process in which molecules are oxidized and the final electron acceptor is an inorganic molecule. Two types of respiration occur, depending on whether an organism is an aerobe or an anaerobe. In Joint initiative of IITs and IISc – Funded by MHRD Page 15 of 73 NPTEL – Biotechnology – Microbiology aerobic respiration – the final electron acceptor is O2 and in anaerobic respiration – it is an inorganic molecule other than O2 or rarely an organic molecule. The Krebs cycle /Citric Acid Cycle/ Tricarboxylic Acid Cycle The pyruvate produced by glycolysis is oxidized completely, generating additional ATP and NADH in the citric acid cycle and by oxidative phosphorylation. However, this can occur only in the presence of oxygen. Oxygen is toxic to organisms that are obligate anaerobes, and are not required by facultative anaerobic organisms. In the absence of oxygen, one of the fermentation pathways occurs in order to regenerate NAD+; lactic acid fermentation is one of these pathways. In eukaryotic cells, the citric acid cycle occurs in the matrix of the mitochondrion. Bacteria also use the TCA cycle to generate energy, but since they lack mitochondria, the reaction sequence is performed in the cytosol with the proton gradient for ATP production being across the plasma membrane rather than the inner membrane of the mitochondrion. Fig. 4. Citric Acid cycle Joint initiative of IITs and IISc – Funded by MHRD Page 16 of 73 NPTEL – Biotechnology – Microbiology • Pyruvic acid, the product of glycolysis, cannot enter the Krebs cycle directly. In a preparatory step; it must lose one molecule of Co2 and become a two-carbon compound. This process is called decarboxylation. The two carbon compound called an acetyl group, attaches to Coenzyme a through a high-energy bond, the resulting complex is known as Acetyl Coenzyme A. During this reaction, pyruvic acid is also oxidized and NAD+ is reduced to NADH. • Oxidation of one glucose molecule produces 2 molecules of pyruvic acid, so for each molecule of glucose, 2 molecules of Co2 are released in the preparatory step, 2 molecules of NADH are produced, and 2 molecules of Acetyl Coenzyme A are formed. • As Acetyl coenzyme A enters the Krebs cycle, CoA detaches from the acetyl group. The two carbon acetyl group combines with a four carbon compound called oxaloacetic acid to form six carbon compound, called citric acid. This synthesis reaction requires energy, which is provided by the cleavage of the high energy bond between the acetyl group and CoA. The formation of citric acid is the first step in the Krebs cycle. • Two decorboxylation reactions take place in the Krebs cycle while converting Isocitric acid to α – Ketoglutaric acid and this to succinyl CoA. • Altogether 3 decarboxylation reactions take place and hence all three carbon atoms in pyruvic acid are eventually released as Co2 by the Krebs cycle. This represents the conversion to Co2 by all 6 carbon atoms contained in the original glucose molecule. • Oxidation-reduction reactions also occurs, where NAD+ and FAD picks up hydrogen atoms to be reduced to NADH and FADH2. • On the whole, for every two molecules of acetyl CoA that enter the cycle, 4 molecules of Co2 and 6 for pyruvic acid are liberated by decorboxylation, 6/8 moelucles of NADH and 2 moleucles of FADH2 are produced by oxidationreduction reactions, and two molecules of ATP are generated by substrate- level phosphorylation. Many of the intermediates in the Krebs cycle also play a role in other pathways, especially in amino acid biosynthesis. • Reduced coenzymes NADH and FADH2 are the important products of the Krebs cycle because they contain most of the energy originally stored in glucose. During Joint initiative of IITs and IISc – Funded by MHRD Page 17 of 73 NPTEL – Biotechnology – Microbiology the next phase of respiration, a series of reductions indirectly transfers the energy stored in those coenzymes to ATP. These reactions are collectively called Electron transport chain. The Electron Transport Chain: • Consists of a sequence of carrier molecules that are capable of oxidation and reduction. • As electrons are passed through the chain, there is a stepwise release of energy, used to drive the chemiosmotic generation of ATP. • In eukaryotic cells, it is contained in the inner membrane of mitochondria. • In prokaryotes, it is found in the plasma membrane. Fig. 5. Electron Transport Chain Three classes of carrier molecules are involved: 1. Flavoproteins – these contain flavin, a coenzyme derived from riboflavin (Vitamin B2). One important flavin coenzyme is flavin mononucleotide (FMN). 2. Cytochromes – proteins with an iron-containing group capable of existing alternately as a reduced form (Fe2+) and an oxidized form (Fe3+). The cytochormes include cytochrome b, C1, a, a3. 3. Ubiquinones or Coenzyme Q – these are small non-protein carriers. Joint initiative of IITs and IISc – Funded by MHRD Page 18 of 73 NPTEL – Biotechnology – Microbiology • Electron transport chains of bacteria are somewhat diverse, and the particular carriers and the order in which they functions may differ from those of other bacteria and from those of eukaryotic mitochondrial systems. Much is known about the electron transport chain in the mitochondria of eukaryotic cells. 1. Transfer of high energy electrons from NADH to FMN, the first carrier in the chain. This transfer involves at the passage of a hydrogen atom with 2e- to FMN, which then pick up an additional H+ from the surrounding aqueous medium. NADH is oxidised to NAD+ and FMN reduced to FMNH2. 2. FMNH2 passes 2H+ to the other side of the mitochondrial membrane and passes 2e- to Q. As a result FMNH2 is oxidized to FMN. Q picks up an additional 2H+ from the medium and releases it on the other side of the membrane. 3. Electrons are passed successively from Q to Cyt b, cyt c1, cyt c, cyt a and cyt a3. Each cytochrome in the chain is reduced as it picks up e-and is oxidised as it gives up electrons. The last cyt a3 passes it electrons to molecular O2, which becomes negatively charged and then picks up protons from the medium to form H2O. • FADH2 adds its electrons to the electron transport chain at a lower level than NADH. Because of this, the electron transport chain produces about one-third less energy for ATP generation when FADH2 donates electrons than when NADH is involved. • FMN and Q accept and release protons as well as electrons and other carrier cytochromes transfer only electrons. • Electron flow down the chain is accompanied at several points by the active transport (Pumping) of protons from the matrix side of the inner mitochondrial membrane to the opposite side of the membrane. The result is build up of protons on one side of the membrane, which provides energy for the generation of ATP by the chemiosmotic mechanism. Chemiosmotic mechanism of ATP generation: • Mechanism of ATP synthesis using the electron transport chain is called chemiosmosis. • Substances diffuse passively across membranes from areas of high concentration to areas of low concentration, this diffusion yields energy. In chemiosmosis, the Joint initiative of IITs and IISc – Funded by MHRD Page 19 of 73 NPTEL – Biotechnology – Microbiology energy released when a substance moves along a gradient is used to synthesize ATP. 1. As energetic electrons from NADH (or chlorophyll) pass down the electron transport chain, some of the carriers in the chain pump actively transport – protons across the membrane. Such carrier molecules are called proton pumps. 2. The phospholipid membrane is normally impermeable to protons, so this onedirectional pumping establishes a proton gradient. The excess H+ on one side of the membrane makes that side positively charged compared with the other side. The resulting electrochemical gradient has potential energy, called the proton motive force. 3. The protons on one side of the membrane can diffuse across the membrane only through special protein channels that contain an enzyme called adenosine triphosphate (ATP synthase). When this flow occurs, energy is released and is used by the enzyme to synthesize ATP from ADP and Pi. • Electron transport chain also operates in photophosphorylation and is located in the thylakoid membrane of cyanobacteria and eukaryotic chloroplasts. Summary of Aerobic respiration: • Electron transport chain regenerates NAD+ and FAD+ which can be used again in glycolysis and Krebs cycle. • Various electron transfers in the electron transport chain generates about 34 molecules of ATP from each molecule of glucose oxidized, 10 NADH and 2 FADH2. • A total of 38 ATP molecules can be generated from one molecule of glucose in prokaryotes. • A total of 36 molecules of ATP are produced in eukaryotes. Some energy is lost when electrons are shuttled across the mitochondrial membranes that separate glycolysis (in the cytoplasm) from the electron transport chain. No such separation exists in prokaryotes. C6H12O6 + 6 CO2 + 38ADP + 38 Pi → 6CO2 + 6H2O + 38 ATP Joint initiative of IITs and IISc – Funded by MHRD Page 20 of 73 NPTEL – Biotechnology – Microbiology Fig. 6. Generation of ATPs and NADH/FADH2 during Aerobic Respiration REFERENCES: Text Books: 1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition). Jones and Bartlett Student edition. 2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An Introduction. Benjamin Cummings. Reference Books: 1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw Hill companies. Joint initiative of IITs and IISc – Funded by MHRD Page 21 of 73 NPTEL – Biotechnology – Microbiology Module 6 – Microbial Metabolism Lecture 3 – Anaerobic respiration and Fermentation Anaerobic Respiration Respiration in some prokaryotes is possible using electron acceptors other than oxygen (O2). This type of respiration in the absence of oxygen is referred to as anaerobic respiration. Electron acceptors used by prokaryotes for respiration or methanogenesis (an analogous type of energy generation in archaea bacteria) are described in the table below. Terminal eAcceptor O2 Reduced End Product H2O NO3 NO2, NH3 or N2 SO4 S or H2S fumarate succinate CO2 CH4 Process aerobic respiration Example Escherichia, Streptomyces Bacillus, Pseudomonas anaerobic respiration: denitrification anaerobic Desulfovibrio respiration: sulfate reduction anaerobic Escherichia respiration: using an organic eacceptor Methanogenesis Methanococcus Biological methanogenesis is the source of methane (natural gas) on the planet. Methane is preserved as a fossil fuel (until we use it all up) because it is produced and stored under anaerobic conditions, and oxygen is needed to oxidize the CH4 molecule. Methanogenesis is not really a form of anaerobic respiration, but it is a type of energy-generating metabolism that requires an outside electron acceptor in the form of CO2. Sulfate reduction is not an alternative to the use of O2 as an electron acceptor. It is an obligatory process that occurs only under anaerobic conditions. Methanogens and sulfate reducers may share habitat, especially in the anaerobic sediments of eutrophic lakes such as Lake Mendota, where they crank out methane and hydrogen sulfide at a surprising rate. Joint initiative of IITs and IISc – Funded by MHRD Page 22 of 73 NPTEL – Biotechnology – Microbiology Nitrate reduction Some microbes are capable of using nitrate as their terminal electron accepter. The ETS used is somewhat similar to aerobic respiration, but the terminal electron transport protein donates its electrons to nitrate instead of oxygen. Nitrate reduction in some species (the best studied being E. coli) is a two electron transfer where nitrate is reduced to nitrite. Electrons flow through the quinone pool and the cytochrome b/c1 complex and then nitrate reductase resulting in the transport of protons across the membrane as discussed earlier for aerobic respiration. N03- + 2e- + 2H+ N02-+ H20 Fig. 7. Nitrate reduction Steps in the dissimilative reduction of nitrate. Some organisms, for example Escherichia coli, can carry out only the first step. All enzymes involved are derepressed by anoxic conditions. Also, some prokaryotes are known that can reduce NO3- to NH4+ in dissimilative metabolism. Denitrification Denitrification is an important process in agriculture because it removes NO3 from the soil. NO3 is a major source of nitrogen fertilizer in agriculture. Almost one-third the cost of some types of agriculture is in nitrate fertilizers. The use of nitrate as a respiratory electron acceptor is usually an alternative to the use of oxygen. Therefore, soil bacteria Joint initiative of IITs and IISc – Funded by MHRD Page 23 of 73 NPTEL – Biotechnology – Microbiology such as Pseudomonas and Bacillus will use O2 as an electron acceptor if it is available, and disregard NO3. This is the rationale in maintaining well-aerated soils by the agricultural practices of plowing and tilling. E. coli will utilize NO3 (as well as fumarate) as a respiratory electron acceptor and so it may be able to continue to respire in the anaerobic intestinal habitat. Nitrite, the product of nitrate reduction, is still a highly oxidized molecule and can accept up to six more electrons before being fully reduced to nitrogen gas. Microbes exist (Paracoccus species, Pseudomonas stutzeri, Pseudomonas aeruginosa, and Rhodobacter sphaeroides are a few examples) that are able to reduce nitrate all the way to nitrogen gas. The process is carefully regulated by the microbe since some of the products of the reduction of nitrate to nitrogen gas are toxic to metabolism. This may explain the large number of genes involved in the process and the limited number of bacteria that are capable of denitrification. Below is the chemical equation for the reduction of nitrate to N2. N03- N02- NO N2O N2 Denitrification takes eight electrons from metabolism and adds them to nitrate to form N2 Fig. 8. Denitification by Pseudomonas stutzeri Four terminal reductases involved in denitrification steps; – Nar: Nitrate reductase (Mo-containing enzyme) – Nir: Nitrite reductase – Nor: Nitric oxide reductase – N2Or: Nitrous oxide reductase Joint initiative of IITs and IISc – Funded by MHRD Page 24 of 73 NPTEL – Biotechnology – Microbiology All can function independently but they operate in unison Fermentation: Fermentation is the process of extracting energy from the oxidation of organic compounds, such as carbohydrates, using an endogenous electron acceptor, which is usually an organic compound. In contrast, respiration is where electrons are donated to an exogenous electron acceptor, such as oxygen, via an electron transport chain. Fermentation is important in anaerobic conditions when there is no oxidative phosphorylation to maintain the production of ATP (adenosine triphosphate) by glycolysis. During fermentation, pyruvate is metabolised to various compounds. Homolactic fermentation is the production of lactic acid from pyruvate; alcoholic fermentation is the conversion of pyruvate into ethanol and carbon dioxide; and heterolactic fermentation is the production of lactic acid as well as other acids and alcohols. Fermentation does not necessarily have to be carried out in an anaerobic environment. For example, even in the presence of abundant oxygen, yeast cells greatly prefer fermentation to oxidative phosphorylation, as long as sugars are readily available for consumption (a phenomenon known as the Crabtree effect). Fig. 9. Respiration and Fermentation pathways Joint initiative of IITs and IISc – Funded by MHRD Page 25 of 73 NPTEL – Biotechnology – Microbiology Lactic acid fermentation is the simplest type of fermentation. In essence, it is a redox reaction. In anaerobic conditions, the cell’s primary mechanism of ATP production is glycolysis. Glycolysis reduces – transfers electrons to – NAD+, forming NADH. However there is a limited supply of NAD+ available in any given cell. • For glycolysis to continue, NADH must be oxidized – have electrons taken away – to regenerate the NAD+ that is used in glycolysis. In an aerobic environment (Oxygen is available), reduction of NADH is usually done through an electron transport chain in a process called oxidative phosphorylation; however, oxidative phosphorylation cannot occur in anaerobic environments (Oxygen is not available) due to the pathways dependence on the terminal electron acceptor of oxygen. • Instead, the NADH donates its extra electrons to the pyruvate molecules formed during glycolysis. Since the NADH has lost electrons, NAD+ regenerates and is again available for glycolysis. Lactic acid, for which this process is named, is formed by the reduction of pyruvate. In heterolactic acid fermentation, one molecule of pyruvate is converted to lactate; the other is converted to ethanol and carbon dioxide. In homolactic acid fermentation, both molecules of pyruvate are converted to lactate. Homolactic acid fermentation is unique because it is one of the only respiration processes to not produce a gas as a byproduct. • Homolactic fermentation breaks down the pyruvate into lactate. It occurs in the muscles of animals when they need energy faster than the blood can supply oxygen. • It also occurs in some kinds of bacteria (such as lactobacilli) and some fungi. It is this type of bacteria that converts lactose into lactic acid in yogurt, giving it its sour taste. These lactic acid bacteria can be classed as homofermentative, where the end-product is mostly lactate, or heterofermentative, where some lactate is further metabolized and results in carbon dioxide, acetate, or other metabolic products. Joint initiative of IITs and IISc – Funded by MHRD Page 26 of 73 NPTEL – Biotechnology – Microbiology C6H12O6 ------- 2 CH3CHOHCOOH. or one molecule of lactose and one molecule of water make four molecules of lactate (as in some yogurts and cheeses): C12H22O11 + H2O ------ 4 CH3CHOHCOOH. In heterolactic fermentation, the reaction proceeds as follows, with one molecule of glucose being converted to one molecule of lactic acid, one molecule of ethanol, and one molecule of carbon dioxide: C6H12O6 -------- CH3CHOHCOOH + C2H5OH + CO2 Before lactic acid fermentation can occur, the molecule of glucose must be split into two molecules of pyruvate. This process is called glycolysis. Fig. 10. Fate of pyruvate in Fermentation Joint initiative of IITs and IISc – Funded by MHRD Page 27 of 73 NPTEL – Biotechnology – Microbiology Mixed fermentations Butanediol Fermentation. Forms mixed acids and gases as above, but, in addition, 2,3 butanediol from the condensation of 2 pyruvate. The use of the pathway decreases acid formation (butanediol is neutral) and causes the formation of a distinctive intermediate, acetoin. Water microbiologists have specific tests to detect low acid and acetoin in order to distinguish non fecal enteric bacteria (butanediol formers, such as Klebsiella and Enterobacter) from fecal enterics (mixed acid fermenters, such as E. coli, Salmonella and Shigella). Butyric acid fermentations, as well as the butanol-acetone fermentation (below), are run by the clostridia, the masters of fermentation. In addition to butyric acid, the clostridia form acetic acid, CO2 and H2 from the fermentation of sugars. Small amounts of ethanol and isopropanol may also be formed. Butanol-acetone fermentation. Butanol and acetone were discovered as the main end products of fermentation by Clostridium acetobutylicum during the World War I. This discovery solved a critical problem of explosives manufacture (acetone is required in the manufacture gunpowder) and is said to have affected the outcome of the War. Acetone was distilled from the fermentation liquor of Clostridium acetobutylicum, which worked out pretty good if you were on our side, because organic chemists hadn't figured out how to synthesize it chemically. You can't run a war without gunpowder, at least you couldn't in those days. Propionic acid fermentation. This is an unusual fermentation carried out by the propionic acid bacteria which include corynebacteria, Propionibacterium and Bifidobacterium. Although sugars can be fermented straight through to propionate, propionic acid bacteria will ferment lactate (the end product of lactic acid fermentation) to acetic acid, CO2 and propionic acid. The formation of propionate is a complex and indirect process involving 5 or 6 reactions. Overall, 3 moles of lactate are converted to 2 moles of propionate + 1 mole of acetate + 1 mole of CO2, and 1 mole of ATP is squeezed out in the process. The propionic acid bacteria are used in the manufacture of Swiss Joint initiative of IITs and IISc – Funded by MHRD Page 28 of 73 NPTEL – Biotechnology – Microbiology cheese, which is distinguished by the distinct flavor of propionate and acetate, and holes caused by entrapment of CO2. REFERENCES: Text Books: 1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition). Jones and Bartlett Student edition. 2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An Introduction. Benjamin Cummings. Reference Books: 1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw Hill companies. Joint initiative of IITs and IISc – Funded by MHRD Page 29 of 73 NPTEL – Biotechnology – Microbiology Module 6 – Microbial Metabolism Lecture 4 –Protein and Lipid Catabolism A. Protein and Amino acid Catabolism Some bacteria and fungi particularly pathogenic, food spoilage and soil microorganisms can use proteins as their source of carbon and energy. 1. Proteases are enzymes that break down proteins into amino acids 2. Amino acids are deaminated, and then enter the Kreb's Cycle. Intact proteins cannot cross bacterial plasma membrane, so bacteria must produce extracellular enzymes called proteases and peptidases that break down the proteins into amino acids, which can enter the cell. Many of the amino acids are used in building bacterial proteins, but some may also be broken down for energy. If this is the way amino acids are used, they are broken down to some form that can enter the Kreb’s cycle. These reactions include: 1. Deamination or Transamination—the amino group is removed or transferred, or converted to an ammonium ion, and excreted. The remaining organic acid (the part of the amino acid molecule that is left after the amino group is removed) can enter the Kreb’s cycle. 2. Decarboxylation—the ---COOH group is removed. 3. Dehydrogenation—a hydrogen is removed. Fig. 11. Process of Transamination Joint initiative of IITs and IISc – Funded by MHRD Page 30 of 73 NPTEL – Biotechnology – Microbiology Fig. 12. Overview of catabolism of Organic Acids Joint initiative of IITs and IISc – Funded by MHRD Page 31 of 73 NPTEL – Biotechnology – Microbiology B. Lipid Catabolism Microorganisms frequently use lipids as energy sources. Triglycerides or triacylglycerols, esters of glycerol and fatty acids, are common energy sources. They can be hydrolyzed to glycerol and fatty acids by microbial lipases. The glycerol is then phosphorylated, oxidized to dihyroxyacetone phosphate, and catabolised in the glycolytic pathway. 1. Lipases are enzymes that break down fats into fatty acid and glycerol components 2. Beta oxidation is the breakdown of fatty acids into two carbon segments (acetyl CoA), Which can enter the Krebs cycle. Functions of lipids in Microbes Lipids are essential to the structure and function of membranes Lipids also function as energy reserves, which can be mobilized as sources of carbon 90% of this lipid is “triacyglycerol” Triacyglycerol-----lipase----->glycerol + 3 fatty acids The major fatty acid metabolism is “β-oxidation” Lipids are broken down into their constituents of glycerol and fatty acids Glycerol is oxidised by glycolysis and the TCA cycle. Bacteria are capable of growth on fatty acids and lipids. Lipids are part of the membranes of living organisms and if available (usually because the organism that was using them dies) can be used as a food source. Lipids are large molecules and cannot be transported across the membrane. A class of extracellular enzymes called lipases are responsible for the breakdown of lipids. Lipases attack the bond between the glycerol molecule oxygen and the fatty acid. Phospholipids are attacked by phospholipases. There are four classes of phospholipases given different names depending upon the bond they cleave. Phospholipases are not particular about their substrate and will attack a glycerol ester linkage containing any length fatty acid attached to it. The result of this Joint initiative of IITs and IISc – Funded by MHRD Page 32 of 73 NPTEL – Biotechnology – Microbiology digestion is a hydrophillic head molecule, glycerol and fatty acids of various chain lengths. The head can be one of several small organic molecules that are funneled into the TCA cycle by one or two reactions that we won't cover here. Glycerol is converted into 3-Phosphoglycerate (depending upon the action of phospholipase C or phospholipase D) and eventually pyruvate via glycolysis. Fig. 13. Lipid Catabolism The β- oxidation pathway Characteristic features; • Every other carbon is converted to a C=O • Allows nucleophilic attack by CoA-SH on remaining chain • 1 CoA is used for every 2 carbon segment to release acetyl-CoA • Each round produces 1 FADH2, 1 NADH, 1 Acetyl-CoA (2 in the last round) Joint initiative of IITs and IISc – Funded by MHRD Page 33 of 73 NPTEL – Biotechnology – Microbiology Step 1: Dehydrogenation of Alkane to Alkene Catalyzed by isoforms of acyl- CoA dehydrogenase (AD) on the inner mitochondrial membrane Step 2: Hydration of Alkene Catalyzed by two isoforms of enoyl-CoA hydratase: Soluble short-chain hydratase (crotonase)Membrane-bound long-chain hydratase, part of trifunctional complexWater adds across the double bond yielding alcohol Step 3: Dehydrogenation of AlcoholCatalyzed by β-hydroxyacyl-CoA dehydrogenase The enzyme uses NAD cofactor as the hydride acceptor Only Lisomers of hydroxyacyl CoA act as substrates Analogous to malate dehydrogenase reaction in the CAC. Fig. 14.The β- oxidation pathway Joint initiative of IITs and IISc – Funded by MHRD Page 34 of 73 NPTEL – Biotechnology – Microbiology Step 4: Transfer of Fatty Acid Chain Catalyzed by acyl-CoA acetyltransferase (thiolase) via covalent mechanism, The carbonyl carbon in β-ketoacyl-CoA is electrophilic Active site thiolate acts as nucleophile and releases acetyl-CoA ; Terminal sulfur in CoA-SH acts as nucleophile. The fatty acid is now two carbons shorter and an Acetyl-CoA, has been generated which can be fed into the TCA cycle. The smaller fatty acid moves through the βoxidation pathway again, producing another Acetyl-CoA and shrinking by 2 carbons. By performing successive rounds of beta oxidation on a fatty acid, it is possible to convert it completely to Acetyl-CoA. Often fatty acids with odd numbers of carbons, the final reaction will yield acetyl-CoA and Coenzyme-A hooked to a three carbon fatty acid (propionyl-CoA). Propionyl-CoA is handled differently by different bacteria. In E. coli it is converted into pyruvate. REFERENCES: Text Books: 1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition). Jones and Bartlett Student edition. 2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An Introduction. Benjamin Cummings. Reference Books: 1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw Hill companies. Joint initiative of IITs and IISc – Funded by MHRD Page 35 of 73 NPTEL – Biotechnology – Microbiology Module 6 – Microbial Metabolism Lecture 5 – Photosynthesis Photosynthesis is the use of light as a source of energy for growth, more specifically the conversion of light energy into chemical energy in the form of ATP. Prokaryotes that can convert light energy into chemical energy include the photosynthetic cyanobacteria, the purple and green bacteria, and the "halobacteria" (actually archaea). The cyanobacteria conduct plant photosynthesis, called oxygenic photosynthesis; the purple and green bacteria conduct bacterial photosynthesis or anoxygenic photosynthesis; the extreme halophilic archaea use a type of nonphotosynthetic photophosphorylation mediated by a pigment, bacteriorhodopsin, to transform light energy into ATP. Net equation: 6CO2+12H2O+LightEnergyC6H12O6+6O2+6H20 Photosynthetic reactions divided into two stages: – Light reaction- light energy absorbed & converted to chemical energy (ATP, NADPH) – Dark reaction-carbohydrates made from CO2 using energy stored in ATP & NADPH Types of bacterial photosynthesis Five photosynthetic groups within domain Bacteria (based on 16S rRNA): 1. Oxygenic Photosynthesis • • Occurs in cyanobacteria and prochlorophytes Synthesis of carbohydrates results in release of molecular O2 and removal of CO2 from • atmoshphere. Occurs in lamellae which house thylakoids containing chlorophyll a/b and phycobilisomes pigments which gather light energy • Uses two photosystems (PS): - PS II- generates a proton-motive force for making ATP. - PS I- generates low potential electrons for reducing power. Joint initiative of IITs and IISc – Funded by MHRD Page 36 of 73 NPTEL – Biotechnology – Microbiology 2. Anoxygenic Photosynthesis • Uses light energy to create organic compounds, and sulfur or fumarate compounds instead of O2. • Occurs in purple bacteria, green sulfur bacteria, green gliding bacteria and heliobacteria. • Uses bacteriochlorophyll pigments instead of chlorophyll. • Uses one photosystem (PS I) to generate ATP in “cyclic” manner. Light Reaction The Light Reactions depend upon the presence of chlorophyll, the primary light-harvesting pigment in the membrane of photosynthetic organisms. The functional components of the photochemical system are light harvesting pigments, a membrane electron transport system, and an ATPase enzyme. The photosynthetic electron transport system of is fundamentally similar to a respiratory ETS, except that there is a low redox electron acceptor (e.g. ferredoxin) at the top (low redox end) of the electron transport chain, that is first reduced by the electron displaced from chlorophyll. There are several types of pigments distributed among various phototrophic organisms. Chlorophyll is the primary light-harvesting pigment in all photosynthetic organisms. Chlorophyll is a tetrapyrrole which contains magnesium at the center of the porphyrin ring. It contains a long hydrophobic side chain that associates with the photosynthetic membrane. Cyanobacteria have chlorophyll a, the same as plants and algae. The chlorophylls of the purple and green bacteria, called bacteriochlorophylls are chemically different than chlorophyll a in their substituent side chains. This is reflected in their light absorption spectra. Chlorophyll a absorbs light in two regions of the spectrum, one around 450nm and the other between 650 -750nm; bacterial chlorophylls absorb from 800-1000nm in the far red region of the spectrum. Carotenoids are always associated with the photosynthetic apparatus. They function as secondary light-harvesting pigments, absorbing light in the blue-green spectral region between 400-550 nm. Carotenoids transfer energy to chlorophyll, at near 100 percent efficiency, from wave lengths of light that are missed by chlorophyll. In addition, carotenoids have an indispensable function to protect the photosynthetic apparatus from photooxidative damage. Carotenoids have long hydrocarbon side chains in a conjugated double bond system. Carotenoids "quench" the powerful oxygen radical, Joint initiative of IITs and IISc – Funded by MHRD Page 37 of 73 NPTEL – Biotechnology – Microbiology singlet oxygen, which is invariably produced in reactions between chlorophyll and O2 (molecular oxygen). Some non-photosynthetic bacterial pathogens, i.e., Staphylococcus aureus, produce carotenoids that protect the cells from lethal oxidations by singlet oxygen in phagocytes. Phycobiliproteins are the major light harvesting pigments of the cyanobacteria. They also occur in some groups of algae. They may be red or blue, absorbing light in the middle of the spectrum between 550 and 650nm. Phycobiliproteins consist of proteins that contain covalently-bound linear tetrapyrroles (phycobilins). They are contained in granules called phycobilisomes that are closely associated with the photosynthetic apparatus. Being closely linked to chlorophyll they can efficiently transfer light energy to chlorophyll at the reaction center. All phototrophic bacteria are capable of performing cyclic photophosphorylation as described above and in Figure 16 and below in Figure 18. This universal mechanism of cyclic photophosphorylation is referred to as Photosystem I. Bacterial photosynthesis uses only Photosystem I (PSI), but the more evolved cyanobacteria, as well as algae and plants, have an additional light-harvesting system called Photosystem II (PSII). Photosystem II is used to reduce Photosystem I when electrons are withdrawn from PSI for CO2 fixation. PSII transfers electrons from H2O and produces O2, as shown in Figure 20. Fig. 15. The cyclical flow of electrons during anoxygenic photosynthesis. Joint initiative of IITs and IISc – Funded by MHRD Page 38 of 73 NPTEL – Biotechnology – Microbiology Fig. 16. Electron flow in oxygenic photosynthesis. Dark reaction The use of RUBP carboxylase and the Calvin cycle is the most common mechanism for CO2 fixation among autotrophs. Indeed, RUBP carboxylase is said to be the most abundant enzyme on the planet (nitrogenase, which fixes N2 is second most abundant). This is the only mechanism of autotrophic CO2 fixation among eucaryotes, and it is used, as well, by all cyanobacteria and purple bacteria. Lithoautotrophic bacteria also use this pathway. But the green bacteria and the methanogens, as well as a few isolated groups of procaryotes, have alternative mechanisms of autotrophic CO2 fixation and do not possess RUBP carboxylase. RUBP carboxylase (ribulose bisphosphate carboxylase) uses ribulose bisphosphate (RUBP) and CO2 as co-substrates. In a complicated reaction the CO2 is "fixed" by addition to the RUBP, which is immediately cleaved into two molecules of 3phosphoglyceric acid (PGA). The fixed CO2 winds up in the -COO group of one of the PGA molecules. Actually, this is the reaction which initiates the Calvin cycle (Fig. 3). The Calvin cycle is concerned with the conversion of PGA to intermediates in glycolysis that can be used for biosynthesis, and with the regeneration of RUBP, the substrate that drives the cycle. After the initial fixation of CO2, 2 PGA are reduced and combined to form hexose-phosphate by reactions which are essentially the reverse of the oxidative Embden-Meyerhof pathway. The hexose phosphate is converted to pentosephosphate, which is phosphorylated to regenerate RUBP. An important function of the Joint initiative of IITs and IISc – Funded by MHRD Page 39 of 73 NPTEL – Biotechnology – Microbiology Calvin cycle is to provide the organic precursors for the biosynthesis of cell material. Intermediates must be constantly withdrawn from the Calvin cycle in order to make cell material. In this regard, the Calvin cycle is an anabolic pathway. The fixation of CO2 to the level of glucose (C6H12O6) requires 18 ATP and 12 NADPH2. Fig. 17. The Calvin cycle and its relationship to the synthesis of cell materials. Most of the phototrophic procaryotes are autotrophs, which mean that they are able to fix CO2 as a sole source of carbon for growth. Just as the oxidation of organic material yields energy, electrons and CO2, in order to build up CO2 to the level of cell material (CH2O), energy (ATP) and electrons (reducing power) are required. The overall reaction for the fixation of CO2 in the Calvin cycle is CO2 + 3ATP + 2NADPH2 ---------> CH2O + 2ADP + 2Pi + 2NADP. The light reactions operate to produce ATP to provide energy for the dark reactions of CO2 fixation. The dark reactions also need reductant (electrons). Usually the provision of electrons is in some way connected to the light reactions. Joint initiative of IITs and IISc – Funded by MHRD Page 40 of 73 NPTEL – Biotechnology – Microbiology Fig. 18. Comparison of electron transport pathways in oxygenic and anoxygenic photosynthesis The differences between plant and bacterial photosynthesis are summarized in Table 3 below. Bacterial photosynthesis is an anoxygenic process. The external electron donor for bacterial photosynthesis is never H2O, and therefore, purple and green bacteria never produce O2 during photosynthesis. Furthermore, bacterial photosynthesis is usually inhibited by O2 and takes place in microaerophilic and anaerobic environments. Bacterial chlorophylls use light at longer wave lengths not utilized in plant photosynthesis, and therefore they do not have to compete with oxygenic phototrophs for light. Bacteria use only cyclic photophosphorylation (Photosystem I) for ATP synthesis and lack a second photosystem. Table 3. Differences between plant and bacterial photosynthesis Plant photosynthesis Bacterial photosynthesis Organisms Plants, algae, cyanobacteria Purple and green bacteria Type of chlorophyll Chlorophyll-a and absorbs 650-750nm bacteriochlorophyll and absorbs 800-1000nm Photosystem I present present (cyclic photophosphorylation) Joint initiative of IITs and IISc – Funded by MHRD Page 41 of 73 NPTEL – Biotechnology – Microbiology present absent Produces O2 yes no Photosynthetic H2O H2S, other sulfur compounds or Photosystem I (noncyclic photophosphorylation) certain organic compounds electron donor Chemosynthesis Chemosynthesis is the biological conversion of one or more carbon molecules (usually carbon dioxide or methane) and nutrients into organic matter using the oxidation of inorganic molecules (e.g. hydrogen gas, hydrogen sulfide) or methane as a source of energy, rather than sunlight, as in photosynthesis. but groups that include conspicuous or biogeochemically-important taxa include the sulfur-oxidizing gamma and epsilon proteobacteria, the Aquificaeles, the Methanogenic archaea and the neutrophilic ironoxidizing bacteria. Chemoautotrophs or lithotrophs, organisms that obtain carbon through chemosynthesis, are phylogenetically diverse, united only by their ability to oxidize an inorganic compound as an energy source. Chemosynthesis runs through the Bacteria and the Archaea. Chemoautotrophs are usually organized into "physiological groups" based on their inorganic substrate for energy production and growth (see Table 2 below). Table 2. Physiological groups of chemoautotrophs Physiological group Energy Oxidized end Organism source product Hydrogen bacteria H2 H2O Alcaligenes, Pseudomonas Methanogens H2 H2O Methanobacterium Carboxydobacteria CO CO2 Rhodospirillum, Azotobacter Nitrifying bacteria* NH3 NO2 Nitrosomonas Nitrifying bacteria* NO2 NO3 Nitrobacter Sulfur oxidizers H2S or S SO4 Thiobacillus, Sulfolobus Iron bacteria Fe ++ Fe+++ Gallionella, Thiobacillus Joint initiative of IITs and IISc – Funded by MHRD Page 42 of 73 NPTEL – Biotechnology – Microbiology *The overall process of nitrification, conversion of NH3 to NO3, requires a consortium of microorganisms. The hydrogen bacteria oxidize H2 (hydrogen gas) as an energy source. The hydrogen bacteria are facultative lithotrophs as evidenced by the pseudomonads that fortuitously possess a hydrogenase enzyme that will oxidize H2 and put the electrons into their respiratory ETS. They will use H2 if they find it in their environment even though they are typically heterotrophic. Indeed, most hydrogen bacteria are nutritionally versatile in their ability to use a wide range of carbon and energy sources. The methanogens used to be considered a major group of hydrogen bacteria until it was discovered that they are Archaea. The methanogens are able to oxidize H2 as a sole source of energy while transferring the electrons from H2 to CO2 in its reduction to methane. Metabolism of the methanogens is absolutely unique, yet methanogens represent the most prevalent and diverse group of Archaea. Methanogens use H2 and CO2 to produce cell material and methane. They have unique enzymes and electron transport processes. Their type of energy generating metabolism is never seen in the Bacteria, and their mechanism of autotrophic CO2 fixation is very rare, except in methanogens. The carboxydobacteria are able to oxidize CO (carbon monoxide) to CO2, using an enzyme CODH (carbon monoxide dehydrogenase). The carboxydobacteria are not obligate CO users, i.e., some are also hydrogen bacteria, and some are phototrophic bacteria. Interestingly, the enzyme CODH used by the carboxydobacteria to oxidize CO to CO2, is used by the methanogens for the reverse reaction - the reduction of CO2 to CO - in their unique pathway of CO2 fixation. The nitrifying bacteria are represented by two genera, Nitrosomonas and Nitrobacter. Together these bacteria can accomplish the oxidation of NH3 to NO3, known as the process of nitrification. No single organism can carry out the whole oxidative process. Nitrosomonas oxidizes ammonia to NO2 and Nitrobacter oxidizes NO2 to NO3. Most of the nitrifying bacteria are obligate lithoautotrophs, the exception being a few strains of Nitrobacter that will utilize acetate. CO2 fixation utilizes RUBP carboxylase and the Calvin Cycle. Nitrifying bacteria grow in environments rich in ammonia, where extensive protein decomposition is taking place. Nitrification in soil and aquatic habitats is an essential part of the nitrogen cycle. Joint initiative of IITs and IISc – Funded by MHRD Page 43 of 73 NPTEL – Biotechnology – Microbiology Chemoautotrophic sulfur oxidizers include both Bacteria (e.g. Thiobacillus) and Archaea (e.g. Sulfolobus). Sulfur oxidizers oxidize H2S (sulfide) or S (elemental sulfur) as a source of energy. Similarly, the purple and green sulfur bacteria oxidize H2S or S as an electron donor for photosynthesis, and use the electrons for CO2 fixation (the dark reaction of photosynthesis). Obligate autotrophy, which is nearly universal among the nitrifiers, is variable among the sulfur oxidizers. Lithoautotrophic sulfur oxidizers are found in environments rich in H2S, such as volcanic hot springs and fumaroles, and deepsea thermal vents. Some are found as symbionts and endosymbionts of higher organisms. Since they can generate energy from an inorganic compound and fix CO2 as autotrophs, they may play a fundamental role in primary production in environments that lack sunlight. As a result of their lithotrophic oxidations, these organisms produce sulfuric acid (SO4), and therefore tend to acidify their own environments. Some of the sulfur oxidizers are acidophiles that will grow at a pH of 1 or less. Some are hyperthermophiles that grow at temperatures of 115°C. Iron bacteria oxidize Fe++ (ferrous iron) to Fe+++ (ferric iron). At least two bacteria probably oxidize Fe++ as a source of energy and/or electrons and are capable of chemoautotrophic growth: the stalked bacterium Gallionella, which forms flocculant rust-colored colonies attached to objects in nature, and Thiobacillus ferrooxidans, which is also a sulfur-oxidizing lithotroph. Fig. 19. Chemoautotrophic or Lithotrophic oxidations. These reactions produce energy for metabolism in the nitrifying and sulfur oxidizing bacteria. Joint initiative of IITs and IISc – Funded by MHRD Page 44 of 73 NPTEL – Biotechnology – Microbiology REFERENCES: Text Books: 1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition). Jones and Bartlett Student edition. 2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An Introduction. Benjamin Cummings. Reference Books: 1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw Hill companies. Joint initiative of IITs and IISc – Funded by MHRD Page 45 of 73 NPTEL – Biotechnology – Microbiology Module 6 – Microbial Metabolism Lecture 6 – Biosynthesis of Amino acids and Lipids Amino acid biosynthesis Amino acid synthesis is the set of biochemical processes (metabolic pathways) by which the various amino acids are produced from other compounds. A fundamental problem for biological systems is to obtain nitrogen in an easily usable form. This problem is solved by certain microorganisms capable of reducing the inert N≡N molecule (nitrogen gas) to two molecules of ammonia in one of the most remarkable reactions in biochemistry. Ammonia is the source of nitrogen for all the amino acids. The carbon backbones come from the glycolytic pathway, the pentose phosphate pathway, or the citric acid cycle. In amino acid production, one encounters an important problem in biosynthesis, namely stereochemical control. Because all amino acids except glycine are chiral, biosynthetic pathways must generate the correct isomer with high fidelity. In each of the 19 pathways for the generation of chiral amino acids, the stereochemistry at the α-carbon atom is established by a transamination reaction that involves pyridoxal phosphate. Almost all the transaminases that catalyze these reactions descend from a common ancestor, illustrating once again that effective solutions to biochemical problems are retained throughout evolution. Amino acid synthesis Amino acids are synthesized from α-ketoacids and later transaminated from another aminoacid, usually Glutamate. The enzyme involved in this reaction is an aminotransferase. α-ketoacid + glutamate ⇄ amino acid + α-ketoglutarate Glutamate itself is formed by amination of α-ketoglutarate: α-ketoglutarate + NH+4 ⇄ glutamate Joint initiative of IITs and IISc – Funded by MHRD Page 46 of 73 NPTEL – Biotechnology – Microbiology Nitrogen fixation: Microorganisms use ATP and a powerful reductant to reduce atmospheric nitrogen to ammonia. Microorganisms use ATP and reduced ferredoxin, a powerful reductant, to reduce N2 to NH3. An iron-molybdenum cluster in nitrogenase deftly catalyzes the fixation of N2, a very inert molecule. Higher organisms consume the fixed nitrogen to synthesize amino acids, nucleotides, and other nitrogen-containing biomolecules. The major points of entry of NH4+ into metabolism are glutamine or glutamate. Nitrifying bacteria N 2 + 8H+ + 8e– + 16ATP + 16 H 2O Nitrogenase 2NH3 + H 2 + 16ADP + 16P i Nitrate Assimilation (Green plants, some fungi and bacteria) Ammonium Assimilation (1) (Carbamyl Phosphate Synthetase) Ammonium Assimilation (2) (Biosynthetic Glutamate Dehydrogenase) and/or (Glutamine Synthetase) Joint initiative of IITs and IISc – Funded by MHRD Page 47 of 73 NPTEL – Biotechnology – Microbiology Glutamate (90%) and Glutamine (10%) are the main sources of organic Nitrogen for microbes. Biosynthesis of some Non-essential Amino Acids (Reactions) 1. Alanine Biosynthesis 2. Aspartate and Asparagine Biosynthesis. Joint initiative of IITs and IISc – Funded by MHRD Page 48 of 73 NPTEL – Biotechnology – Microbiology 3. Proline Biosynthesis Amino acids are made from intermediates of the citric acid cycle and other major pathways Glutamate dehydrogenase catalyzes the reductive amination of α-ketoglutarate to glutamate. A transamination reaction takes place in the synthesis of most amino acids. At this step, the chirality of the amino acid is established. Alanine and aspartate are synthesized by the transamination of pyruvate and oxaloacetate, respectively. Glutamine is synthesized from NH4+ and glutamate, and asparagine is synthesized similarly. Proline and arginine are derived from glutamate. Serine, formed from 3phosphoglycerate, is the precursor of glycine and cysteine. Tyrosine is synthesized by the hydroxylation of phenylalanine, an essential amino acid. The pathways for the biosynthesis of essential amino acids are much more complex than those for the nonessential ones. Activated Tetrahydrofolate, a carrier of one-carbon units, plays an important role in the metabolism of amino acids and nucleotides. This coenzyme carries one-carbon units at three oxidation states, which are interconvertible: most reduced— methyl; intermediate—methylene; and most oxidized—formyl, formimino, and methenyl. Joint initiative of IITs and IISc – Funded by MHRD Page 49 of 73 NPTEL – Biotechnology – Microbiology Fatty Acid/ Lipid Biosynthesis Fatty acid biosynthesis occurs in following phases; 1. Synthesis of malonyl-CoA via Acetyl-CoA Carboxylase 2. Fatty Acid Synthase 3. Fatty acid elongation and desaturation Site: Synthesis of fatty acids takes place in the cytoplasm and involves initiation of synthesis by the formation of acetoacetyl-ACP and then an elongation cycle where 2 carbon units are successively added to the growing chain. Acyl carrier protein (ACP) serves as a chaperone for the synthesis of fatty acids. The growing fatty acid chain is covalently bound to ACP during the entire synthesis of the fatty acid and only leaves the protein when it is attached to the glycerol backbone of the forming lipid. ACP is one of the most abundant proteins in the bacterial cell (60,000 molecules per E. coli cell) which makes sense given the amount of lipid that must be synthesized to make an entire cell membrane. The formation of acetoacetyl-ACP can be catalyzed by a number of enzymes, but in all cases the starting substrate is acetyl-CoA. Once formed, acetoacetyl-ACP enters the elongation cycle for fatty acid synthesis. This cycle is the reverse of the β-oxidation of fatty acids discussed earlier. The first step in the elongation cycle is condensation of malonyl-CoA with a growing acetoacetyl-ACP chain. This adds two carbons to the chain. The next three reactions use 2 NADPH to reduce the β-ketone and generate an acyl-ACP molecule two carbons longer than the original substrate. The acyl-ACP molecule continues through the cycle until the appropriate chain length is reached. In E. coli fatty acid chains in lipids are 12-20 carbons long. The length of the fatty acid chains and the number of double bonds (unsaturation) is dependent upon the temperature the bacteria are growing at. The membrane must remain fluid. Using short chain fatty acids with higher degrees of unsaturation increases the fluidity of the membrane. As the temperature increases, longer fatty acid chains with fewer double bonds will be more prevalent in the membrane. The input to fatty acid synthesis is acetyl-CoA, which is carboxylated to malonylCoA. Joint initiative of IITs and IISc – Funded by MHRD Page 50 of 73 NPTEL – Biotechnology – Microbiology The ATP-dependent carboxylation provides energy input. The CO2 is lost later during condensation with the growing fatty acid. The spontaneous decarboxylation drives the condensation. Acetyl-CoA Carboxylase catalyzes the 2-step reaction by which acetyl-CoA is carboxylated to form malonyl-CoA. As with other carboxylation reactions (e.g., Pyruvate Carboxylase), the enzyme prosthetic group is biotin. ATP-dependent carboxylation of the biotin, carried out at one active site (1), is followed by transfer of the carboxyl group to acetyl-CoA at a second active site (2). The overall reaction, which is is spontaneous, may be summarized as: HCO3- + ATP + acetyl-CoA -------- ADP + Pi + malonyl-CoA Garrett & Grisham; Biochemistry, 2/e Fig. 20. The Acyl Carrier protein (ACP) Joint initiative of IITs and IISc – Funded by MHRD Page 51 of 73 NPTEL – Biotechnology – Microbiology Garrett & Grisham; Biochemistry, 2/e Fig. 21. Fatty acid synthesis Joint initiative of IITs and IISc – Funded by MHRD Page 52 of 73 NPTEL – Biotechnology – Microbiology Fig. 22. Synthesis of palmitic acid The overall synthesis of palmitic acid: The fatty acyl chain grows by two-carbon units donated by activated malonate, with loss of CO2 at each step. The initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. After each two-carbon addition, reductions convert the growing chain to a saturated fatty acid of four, then six, then eight carbons, and so on. The final product is palmitate (16:0). Stages of Fatty acid synthesis • Overall goal is to attach a two-carbon acetate unit from malonyl-CoA to a growing chain and then reduce it. • Reaction involves cycles of four enzyme-catalyzed steps – Condensation of the growing chain with activated acetate. – Reduction of carbonyl to hydroxyl. – Dehydration of alcohol to trans-alkene. – Reduction of alkene to alkane. • The growing chain is initially attached to the enzyme via a thioester linkage • During condensation, the growing chain is transferred to the acyl carrier protein • After the second reduction step, the elongated chain is transferred back to fatty acid synthase Joint initiative of IITs and IISc – Funded by MHRD Page 53 of 73 NPTEL – Biotechnology – Microbiology Lehninger Principles of Biochemistry, Fifth Edition Fig. 23. Stages of fatty aid synthesis Addition of two carbons to a growing fatty acyl chain: a four-step sequence. Each malonyl group and acetyl (or longer acyl) group is activated by a thioester that links it to fatty acid synthase, a multienzyme system. 1. Condensation of an activated acyl group (an acetyl group from acetyl-CoA is the first acyl group) and two carbons derived from malonyl-CoA, with elimination of CO2 from the malonyl group, extends the acyl chain by two carbons. The β-keto product of this condensation is then reduced in three more steps nearly identical to the reactions of β oxidation, but in the reverse sequence: 2. The β-keto group is reduced to an alcohol, Joint initiative of IITs and IISc – Funded by MHRD Page 54 of 73 NPTEL – Biotechnology – Microbiology Lehninger Principles of Biochemistry, Fifth Edition Fig. 24. Stages of fatty aid synthesis Addition of two carbons to a growing fatty acyl chain: a four-step sequence. Each malonyl group and acetyl (or longer acyl) group is activated by a thioester that links it to fatty acid synthase, a multienzyme system described later in the text. 1 Condensation of an activated acyl group (an acetyl group from acetyl-CoA is the first acyl group) and two carbons derived from malonyl-CoA, with elimination of CO2 from the malonyl group, extends the acyl chain by two carbons. The mechanism of the first step of this reaction is given to illustrate the role of decarboxylation in facilitating condensation. The β-keto product of this condensation is then reduced in three more steps nearly identical to the reactions of β oxidation, but in the reverse sequence: 2 the β-keto group is reduced to an alcohol, 3 elimination of H2O creates a double bond, and 4 the double bond is reduced to form the corresponding saturated fatty acyl group. Joint initiative of IITs and IISc – Funded by MHRD Page 55 of 73 NPTEL – Biotechnology – Microbiology Importance in organisms; Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, and K), monoglycerides, diglycerides, triglycerides, phospholipids, and others. The main biological functions of lipids include energy storage, as structural components of cell membranes, and as important signaling molecules. REFERENCES: Text Books: 1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition). Jones and Bartlett Student edition. 2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An Introduction. Benjamin Cummings. Reference Books: 1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw Hill companies. Joint initiative of IITs and IISc – Funded by MHRD Page 56 of 73 NPTEL – Biotechnology – Microbiology Module 6 - Metabolism Lecture – 7 - Biosynthesis of Pyramidines 1. INTRODUCTION Nucleotides are essential for many cellular functions, including the storage of genetic information, gene expression, energy metabolism, cell signaling, and biosynthesis. In a cell, nucleotides exist primarily as 5'-triphosphates. ATP is the most prevalent nucleotide, reaching mM concentrations in many cell types, while other nucleotides may be present at much lower concentrations (cAMP). Biological significance of nucleotides 1. Building blocks of nucleic acids (DNA and RNA). 2. Involved in energy storage, muscle contraction, active transport, and maintenance of ion gradients. 3. Activated intermediates in biosynthesis (e.g. UDP-glucose, S-adenosylmethionine (SAM). 4. Components of coenzymes (NAD+, NADP+, FAD, FMN, and CoA) 5. Metabolic regulators: a. Second messengers (cAMP, cGMP) b. Phosphate (PO32- ) donors for phosphorylation of kinases and phosphatases in signal transduction (ATP) c. Regulation of some enzymes via adenylation and uridylylation Each nucleotide contains a purine or pyrimidine base, a ribose or deoxyribose sugar, and a phosphate: Nucleotide Purine or Pyrimidine Base O 5' -O P O O O- H 4' 3' H O Phosphate H 1' 2' H H(OH) Pentose sugar Nucleoside FIG. 25. STRCUTURE OF NUCLEOTIDE Joint initiative of IITs and IISc – Funded by MHRD Page 57 of 73 NPTEL – Biotechnology – Microbiology Sources of nucleotides in a cell: 1) Degradation of nucleic acids (salvage pathways). Free purine bases can be recycled by coupling with the ribose phosphate moiety, 5-phospho-ribosyl-1-pyrophosphate (PRPP), to form nucleotide monophosphates: ad enine p hosph oribosyl transfera se Adenine + PRPP Adenylate + PPi Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) Guanine + PRPP Hypoxanthine + PRPP Guanylate + PPi Inosinate+ PPi FIG. 26. SORUCES OF NUCLEOTIDES (BENJAMIN CUMMINGS, 2008) Joint initiative of IITs and IISc – Funded by MHRD Page 58 of 73 NPTEL – Biotechnology – Microbiology DE NOVO SYNTHESIS OF PYRIMIDINES Metabolic precursors of nucleotides include amino acids, CO2, and ribose-5-phosphate. Sources of carbon and nitrogen atoms in pyrimidine ring from carbamoyl phosphate N3 C NH2 C 4 2 1 N 5C from aspartate O 6C -O C CH2 O O CH H2N PO32- COO- O3PO CH2 O O -O -O P P O OH 5-phosphoribosyl-1-pyrophosphate (PRPP) is the source of phosphoribose OO O OH Fig. 27. Soruces of carbon and nitrogen atoms in pyrimidine ring (Stryer’s Biochemistry 4th edition) The pyrimidine ring is synthesized in a 6-step process that requires participation of several enzymes. Most required enzymes are cytosolic, with the exception of orotate dehydrogenase that is localized in mitochondria (see below). The general strategy is to use pre-assembled components (carbamoyl phosphate and aspartate) to make a pyrimidine ring which is then attached to the phosphoribose. Part 1. The formation of carbamoyl phosphate is catalyzed by cytosolic carbamoyl phosphate synthetase II (CPS). ATP ADP HO (from Gln) NH3 O O HO C C C CPS O phosphorylation of bicarbonate P O ATP ADP Carbamic acid O-O P C CPS NH2 NH2 O- ammonia displaces the phosphate OCarboxyphosphate Bicarbonate HO O HO O CPS O Pi O O C O phosphorylation Carbamic acid NH2 Carbamoyl phosphate Fig. 28. The formation of carbamoyl phosphate Joint initiative of IITs and IISc – Funded by MHRD Page 59 of 73 NPTEL – Biotechnology – Microbiology Regulated Step in Pyrimidine Biosynthesis General Mechanism for replacing carbonyl oxygen with amino group: The ammonia necessary for the formation of carbamic acid originates from glutamine: NH2 H2N H2 C C O NH2 CH C H2 OH CPS C O Glutamine (Gln) NH3 H2 C HO C CH C H2 OH C O O glutamate Note that a single enzyme, carbamoyl phosphate synthetase II, catalyzes all 4 reactions required for the synthesis of carbamoyl phosphate. Reaction intermediates are channeled internally between the active sites responsible for phosphorylation, carbamic acid formation, and glutamine hydrolysis, without dissociating from the enzyme. This is necessary to protect unstable intermediates from hydrolysis and to drive the reactions forward. Joint initiative of IITs and IISc – Funded by MHRD Page 60 of 73 NPTEL – Biotechnology – Microbiology Part 2. The formation of orotate The committed step in the biosynthesis of pyrimidines is the formation of N-carbamoylaspartate from aspartate and carbamoyl phosphate. Carbamoyl aspartate is then cyclized and oxidized by NAD+ to orotate: O Aspartate Transcarbamoylase Asp O-O Pi HN O O P C O NH2 + C OOC Asp replaces Pi CH H COO cyclization C H2 Carbamoyl phosphate H2O NH2 Dihydroorotate Dehydrogenase O Dihydroorotase HN OOC N-Carbamoylaspartate CH CH C H2 NADH + H C HN C NH C OOC O C H2 C C H oxidation O Orotate Dihydroorotate Electrons are channeled directly to the transport chain of mitochondria NH2 -OOC + + NAD NH C O COO- Aspartate Fig. 29. Formation of Orotate (Stryer's Biochemistry 5th ed) Note: 1. Aspartate transcarbamoylase is allosterically inhibited by CTP, the final product of pyrimidine nucleotide biosynthesis. This ensures a tight control of CTP concentrations in a cell. • N-(phosphonacetyl)-L-aspartate (PALA) is a bisubstrate analog inhibitor of aspartate transcarbamoylase. It resembles carbamoylphosphate+ aspartate • Human carbamoyl phosphate synthetase and aspartate transcarbamoylase are part of the same protein. O O - C HN OOC O3PO CH2 NH C O -O Orotate O O OH PPi -O P C C H O CH O3PO C O OCH2 P O - C HN pyrimidine phosphoribosyltransferase N O COO O OH 5-phosphoribosyl-1-pyrophosphate (PRPP) OH OH Orotidylate Joint initiative of IITs and IISc – Funded by MHRD C Page 61 of 73 NPTEL – Biotechnology – Microbiology Part 3: Formation of UMP. Orotate coupling to ribose in the form of 5-phosphoribosyl1-pyrophosphate (PRPP) produces orotidylate (OMP): PRPP is produced by phosphorylation of ribose-5-phosphate (from pentose phosphate pathway): 2- ATP O3PO CH2 AMP 2- O3PO CH2 O O OH -O P PRPP synthetase OH O -O P O OH OH ribose-5-phosphate O- O O OH 5-phosphoribosyl-1-pyrophosphate (PRPP) Decarboxylation of orotidylate yields uridylate (UMP), a major pyrimidine nucleotide. O C HN - Phosp O CH O3PO C O CH2 N O UMP H+ CO2 - CH O3PO C C O CH2 COO horylat ion of C HN N O C H UMP synthase OH OH OH Orotidylate OH Uridylate (UMP) by kinases gives rise to UDP and UTP: UMP kinase UMP + ATP UDP + ADP nucleotide diphosphate kinase UTP + ADP UDP + ATP Joint initiative of IITs and IISc – Funded by MHRD Page 62 of 73 NPTEL – Biotechnology – Microbiology Formation of cytosine nucleotides Cytidine triphosphate is formed from uridine triphosphate by the replacement of a carbonyl group with an amino group. This is accomplished by a mechanism similar to carbamoyl phosphate synthesis (p. 4). The O4 of the uridine is phosphorylated, followed by displacement with ammonia. CTP synthetase is feedback-inhibited by CTP. Gln + H2O NH2 O Glu C HN 4- NH3 CH O3PO3PO3PO-H2C CH O3PO3PO3PO-H2C N O CH2 C N O C H H ATP ADP + Pi OH OH C O C O CH2 4- C N OH OH CTP UTP Feedback Inhibition in Bacteria Pyrimidine Biosynthesis Nucleotide concentrations and their biosynthesis are highly regulated. This tight control is achieved by allosteric modulation (nucleotide end products act as negative effectors). Glutamine + HCO3- + ATP PRPP Carbamoyl phosphate OMP UMP UDP UTP CTP Fig. 30. Feedback inhibition in bacterial pyrimidine biosynthesis Examples of feedback inhibition: Carbamoyl phosphate synthetase is feedback – inhibited by CTP . Aspartate transcarbamoylase (ATCase) is inhibited by CTP. Joint initiative of IITs and IISc – Funded by MHRD Page 63 of 73 NPTEL – Biotechnology – Microbiology Furthermore, the concentrations of key enzymes are transcriptionally regulated during the cell cycle, with increased rates of nucleotide synthesis in the late G1/early S phase of the cell cycle preceding DNA replication. Orotic acid urea: a rare disease associated with defects in pyrimidine biosynthesis – Symptoms: anemia, growth retardation, orotic acid excretion in urine. – Causes: a defect in phosphoribosyl transferase or orotidylate decarboxylase – Treatment: patients are fed uridine: U → UMP → UDP → UTP UTP inhibits CPS II (regulated step), preventing the biosynthesis of orotic acid Pyrimidine biosynthesis: Take home message 1. Pyrimidines are synthesized using both de novo and salvage pathways. 2. The pyrimidine ring is synthesized from pre-assembled ingredients (carbamoyl phosphate and aspartate) and then attached to ribose. 3. Pyrimidine biosynthesis is tightly regulated via feedback inhibition (CTP synthetase, ATCase) and transcriptional regulation (ATCase). 4. Most of the necessary enzymes are located in the cytosol with the exception of dihydroorotate (localized in the mitochondria). 5. The mammalian enzymes are multifunctional, e.g. CAD (three activities), UMP synthetase (two activities). REFERENCES: Text Books: 1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition). Jones and Bartlett Student edition. 2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An Introduction. Benjamin Cummings. Reference Books: 1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw Hill companies. Joint initiative of IITs and IISc – Funded by MHRD Page 64 of 73 NPTEL – Biotechnology – Microbiology Module 6 - Metabolism Lecture – 8 - Biosynthesis of Purines and Peptidoglycan DE NOVO SYNTHESIS OF PURINES Sources of carbon and nitrogen atoms in purine ring: Gly (2) CO2 (5) 6 Aspartate (6) amino 1N C 7 5 N 8 C C 2C C N N10-THF (7) N 4 3 (the order of incorporation) N10-THF (3) 9 Glutamine amide (1) Glutamine amide (4) Fig. 31. Sources of carbon and nitrogen in purine ring (Stryer's Biochemistry 5th ed) General features: 1. Unlike pyrimidines that do not acquire the ribose until the base moiety is synthesized, purine ring is assembled on PRPP. 2. IMP is synthesized first and serves as a precursor to adenine and guanine nucleotides. Step 1: Formation of 5-phosphoribosyl-1-amine from PRPP (committed step) by the action of glutamine phosphoribosyl amidotransferase. PRPP provides the foundation for purine biosynthesis. Glutamine phosphoribosyl amidotransferase has two domains: one hydrolyzes glutamine to ammonia, and the other one is phosphoribosyl transferase domain. Ammonia is channeled to site II without leaving the enzyme. Glu + NH3 Gln + H2O 2- Future N-9 of the purine ring 2- O3PO O3PO CH2 CH2 O -O O -O P O OH β-configuration of the sugar OP O NH2 O PPi is replaced by NH2 O inversion of configuration OH 5-phosphoribosyl-1-pyrophosphate (PRPP) Joint initiative of IITs and IISc – Funded by MHRD PPi OH OH 5-Phosphoribosyl-1-amine Page 65 of 73 NPTEL – Biotechnology – Microbiology This is the committed step. It is feedback inhibited by IMP. GMP, AMP Step 2: Glycine is coupled to the amino group of phosphoribosylamine glycinamide ribonucleotide synthetase ATP + Gly N7 ADP + Pi C4 C5 NH P-ribose P-ribose-NH2 phorphoribosyl amine O C CH2 CH2 C O Gly is coupled to the amino group - NH3+ glycinamide ribonucleotide NH3+ O This is the only step in purine biosynthesis that contributes more than one atom to the purineskeleton. a) The carboxylate group of Gly is activated by phosphorylation to form acylphosphate. b) The phosphate is then displaced by the amino group of phosphoribosylamine. O O -O P OH -O P O NH3+ C OP-ribose-NH2 O- P-ribose-NH CH2 NH3+ C O CH2 O Step 3: Transfer of a formyl group from N10-formyltetrahydrofolate to the amino group of the glycine residue (catalyzed by glycinamide ribonucleotide transferase). N10-formyltetrahydrofolate (THF) serves as a C1 carbon donor. O N10-formylNH3+ P-ribose NH C O Glycinamide ribonucleotide THF H THF CH2 P-ribose α -amino terminus is formylated NH C C4 C C8 NH N7 H2 N H N N N N H CH2 C5 OH N9 O Formylglycinamide ribonucleotide CH2 5 O C H CH2 O N C 10 O- O C N H H2 H2 O CH C C C OH H 10 N -formyl -tetrahydrofolate (THF) Joint initiative of IITs and IISc – Funded by MHRD Page 66 of 73 NPTEL – Biotechnology – Microbiology Step 4: The inner amide group is converted to an amidine by the replacement with ammonia derived from glutamine: O O C H P-ribose NH ATP H ADP + Pi NH C C C8 NH N7 N9 CH2 NH P-ribose Glu + NH3 Gln + H2O O CH2 C5 N H Formylglycinamidine ribonucleotide N3 Formylglycinamide ribonucleotide C4 C =NH replaces =O Note: Steps 1-4 are catalyzed by a multienzyme complex. Many of the intermediates in purine biosynthesis are unstable in aqueous solution and only exist in solvent protected environment. The formation of multienzyme complexes makes it possible to internally channel the product of one reaction to the next catalytic center without solvent exposure. Step 5: An intramolecular coupling reaction accompanied by a loss of water forms the five-membered imidazole ring. The carbonyl is activated by phosphorylation and the phosphate is displaced by an amino group as shown on p. 5 (top) O AIR synthetase C H NH ATP C8 ADP + Pi HC N7 N CH C5 P-ribose NH C CH2 N - H2O ring closure H Formylglycinamidine ribonucleotide Joint initiative of IITs and IISc – Funded by MHRD P-ribose N N9 C C4 N3 NH2 5-aminoimidazole ribonucleotide Page 67 of 73 NPTEL – Biotechnology – Microbiology Step 6: Bicarbonate adds first to the exocyclic amino group and then is transferred to the neighboring carbon atom of the imidazole ring:Enzyme: AIR carboxylase ATP + O HO N N HC O- CH P-ribose HC - H2O NH2 8 carboxyl transfer HC CH O C N C N ADP + Pi N 7 O 5 C 9N 6 O- C 4 HN P-ribose carboxylation of the amino group P-ribose O- NH2 Carboxyaminoimidazole ribonucleotide 5-aminoimidazole ribonucleotide Step 7: The imidazole carboxylate is phosphorylated and the phosphate is displaced by the amino group of aspartate: ATP O N ADP + Pi HC C C O- C N P-ribose - H2O N P-ribose NH2 H2N Carboxyaminoimidazole ribonucleotide CH CO2- N H C CH CO2CH2 NH2 CO2- 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide CH2 Asp O N HC CO2- Enzyme: SAICAR synthetase Step 8. Elimination of fumarate is catalyzed by lyase. The carbon skeleton of aspartate is lost as fumarate (only the amino group is contributed by aspartate). Enzyme: adenylsuccinate lyase CO2- CH HC O N HC N P-ribose N H NH2 CO2- CH HC CH H CO2- 5-amidoimidazole-4-(N-succinylcarboxxamide) ribonucleotide Joint initiative of IITs and IISc – Funded by MHRD O N CO2- C C fumarate N P-ribose C NH2 C NH2 5-aminoimidazole-4-carboxamide ribonu Page 68 of 73 NPTEL – Biotechnology – Microbiology Step 9: The second formyl group is added from N10-formyltetrahydrofolate: O N HC 10 N -formylTHF C HC THF C NH2 C N O N P-ribose NH2 NH2 C N P-ribose NH 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) H C C2 O 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) Enzyme: AICAR transformylase Step 10: Cyclization/dehydration produces inosinate: 7 O N HC 8 N P-ribose NH2 C NH 9 C 4 6 C P-ribose H C N O 5 HC AMP cyclohydrolase C N 3 NH 1 N CH 2 H 2O O Inosinate (IMP) 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) Formation of AMP and GMP from IMP IMP is a common precursor to both AMP and GMP. 1. Formation of adenylate (AMP): the C-6 carbonyl group of inosinate is replaced with the amino group from Asp. O- Adenylsuccinate synthetase O CH GTP O Asp NH C N N - H2O N P-ribose N P-ribose N (IMP) Asp = CH C O- H COONH2 N O ON N - H2O P-ribose N Adenylsuccinate Adenylate (AMP) CH2 C H lyase O H2N Inosinate H fumarate CH HN N GDP + Pi N -OOC C O OH Note that GTP is used as a phosphate donor instead of ATP Joint initiative of IITs and IISc – Funded by MHRD Page 69 of 73 NPTEL – Biotechnology – Microbiology 2. Formation of guanylate (GMP). Inosinate is first oxidized to xanthylate, and the C2 carbonyl is then converted to an amino group: IMP dehydrogenase GMP synthetase NAD+ O H2O N O O N NADH + H+ ATP AMP + PPi NH NH NH N N N N N N H P-ribose P-ribose O H3N + Glu N H2O + Gln P-ribose Xanthylate Inosinate NH2 Guanylate (GMP) (XMP) (IMP) Regulation of Purine Biosynthesis: • AMP, GMP and IMP are feedback inhibitors of purine biosynthesis • Purine biosynthesis is energetically expensive (6 moles ATP used per 1 mol IMP) • Nucleotide concentrations are tightly regulated in a cell. Loss of regulation may lead to clinical disease (e.g. gout). 1. PRPP synthesis is inhibited by AMP, IMP, GMP (indicators of poor energy status) 2. The committed step (phosphoribosylamine formation) is inhibited by all purine mononucleotides. 3. AMP and GMP inhibit their own synthesis from IMP nucleotide Reciprocal substrate regulation balances the relative purine levels: AMP synthesis requires GTP as the energy source GMP synthesis requires ATP as the energy source Fig. 32. Regulation of purine biosynthesis Joint initiative of IITs and IISc – Funded by MHRD Page 70 of 73 NPTEL – Biotechnology – Microbiology Biosynthesis of NAD+ Nicotinamide adenine dinucleotide (NAD+) and its phosphorylated analog, NADP+, are important coenzymes that participate in a number of biological processes involving electron transfer. NAD+ contains an AMP moiety as part of the molecule: NAD+ is a two electron acceptor (H-) H O O Nicotinamide O H H NH2 NH2 NH2 O P -O P N O N NH2 OH N OH N P P O R N O -O N O R NAD+ Adenine NADH N O Ribose O OH OH NAD+ NAD+ synthesis requires nicotinate (vitamin B6), which is derived from tryptophan. In the first step, nicotinate ribonucleotide is formed from nicotinate and PRPP: COOPRPP PPi COO- N 2- O3PO O N H Nicotinate H H OH H OH H Ribose-P Nicotinate ribonucleotide In the following steps, an AMP moiety is transferred from ATP to nicotinate ribonucleotide to form desamido-NAD+. Finally, the carboxyl group of desamido-NAD is converted to amide using glutamine as an ammonia donor: Joint initiative of IITs and IISc – Funded by MHRD Page 71 of 73 NPTEL – Biotechnology – Microbiology NADP is obtained by phosphorylation of the 2'-OH of the adenine ribose by ATP in the presence of NAD+ kinase. Purine biosynthesis: 1. Purines are synthesized using both de novo and salvage pathways. 2. The purine ring is built on a ribose skeleton to make IMP, followed by branches to AMP and GMP. Amino acids serve as N donors, while CO2 and C1 units of N10-formylTHF are carbon donors. 3. The general mechanism involves ATP-mediated phosphorylation of carbonyl oxygen, followed by phosphate displacement by an amine. 4. Purine biosynthesis is tightly regulated via feedback inhibition. Formation of phosphoribosylamine is the committed step. The loss of regulation can lead to a clinical disease. 5. NAD+ is biosynthesized from nicotinate, ATP, and PRPP. Glutamine is used as an amino donor. PEPTIDOGLYCAN SYNTHESIS: Bacterial cell walls contain a large, complex peptidoglycan molecule consisting of log polysaccharide chains made of alternating N-acetykmuramic acid (NAM) and Nacetylglucosamine (NAG) residues. Pentapeptide chains are attached to the NAM groups. The polysaccharide chains are connected through their pentapeptides or by interbridges. Peptidoglycan synthesis is a multistep process that has been best studied in the grampositive bacterium Staphylococcus aureus. Two carriers participate: uridine diphosphate (UDP) and bactoprenol. Bactoprenol is a 55-carbon alcohol that attaches to NAM by a pyrophosphate group and moves peptodoglycan components through the hydrophobic membrane. Transpeptidation is an important reaction, wherein finally peptide cross-links between peptidoglycan chains are formed. Peptidoglycan synthesis is particularly vulnerable to disruption by antimicrobial agents. Inhibition at any stage can weaken the cell wall and can lead to osmotic lysis. Many antibiotics interfere with peptidoglycan synthesis. For example, Penicillin inhibits the transpeptidation reaction and bacitracin blocks the dephosphorylation of bactoprenol pyrophosphate. Joint initiative of IITs and IISc – Funded by MHRD Page 72 of 73 NPTEL – Biotechnology – Microbiology PATTERNS OF CELL WALL FORMATION: To grow and divide efficiently, a bacterial cell must add new peptidoglycan to its cell wall in a precise and well-regulated way while maintaining wall shape and integrity in the presence of high osmotic pressure. The growing bacterium must be able to degrade it just enough to provide accept ends for the incorporation of new peptidoglycan units. It must also reorganize peptidoglycan structure when necessary. This limited peptidoglycan digestion is accomplished by enzymes known as autolysins, some of which attack the polysaccharide chains, while others hydrolyze the peptide cross-links. Autolysin inhibitors keep the activity of these enzymes under tight control. There seems to be two general patterns of location and distribution of cell wall synthetic activity and varies with species. Many gram-positive cocci have only one to a few zones of growth. The principal growth zone is usually at the site of septum formation, and new cell wall halves are synthesized back-to-back. The second pattern of synthesis occurs in the rod shaped bacterial and occurs at the site of septum formation just as before, but growth sites also are scattered along the cylindrical portion of the rod. Thus growth is distributed more diffusely in rod-shaped bacteria than in the cocci. Synthesis must lengthen rod-shaped cells as well as divide them. Presumably this accounts for the differences in wall formation pattern. REFERENCES: Text Books: 1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition). Jones and Bartlett Student edition. 2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An Introduction. Benjamin Cummings. Reference Books: 1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw Hill companies. Joint initiative of IITs and IISc – Funded by MHRD Page 73 of 73