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Enterics Emphasize novel pyruvate enzymes Example of free radicals involved in C-C bond cleavage. Gram negative bacteria that ferment sugars to acids and gas. All use glycolysis Mixed acid group: Escherichia, Salmonella, Shigella, Proteus. Make lactic, acetic, succinic, formic acids Butanediol fermentors: Enterobacter, Serratia, Erwinia Mixed acid at neutral pH Make 2,3-butanediol at low pH Ways to cleave a C-C bond Carbonyl beta to another oxygen-containing molecule Alpha decarboxylation: use TPP to stabilize carbanion Rearrangements: use free radical mechanism, free radical provided by 5deoxyadenosyl on B12 Other free radicals can be formed on proteins: glycyl and tyrosyl free radicals See how E. coli cleaves pyruvate by free radical mechanism Fermentation Products Products E. col i For m ate Acetate La ctate Su ccini c Ethanol Butandiol C O2 H2 2.4 36.5 79.5 10.7 49.8 0.3 88 75 E. aerogenes 17 0.5 2.9 69.5 66.4 172 35.4 Mixed acid fermentation Glucose to pyruvate by glycolysis New enzymes Pyruvate metabolism by pyruvate-formate lyase Pyruvate + CoA --> acetyl-CoA + formate (HCOOH) No TPP (glycine free radical), no NADH made Still get high energy intermediate, but don’t have to recycle NADH Mechanism of Pyruvate-formate lyase Free radical cleavage of C-C bond Transfer stable free radical on glycine to one of the sulfurs in cysteine at the active site. cys418 cys419 S• HS O C CH3 C cys419 cys418 S HS •O C CH3 O Ocys418 C O cys418 cys419 S• HS O CH3-C-S-CoA O O- C CH3 cys419 S HS C S HS CoA-S• CoA cys418 O C CH3 O O• HCOO- cys419 O C CH3 cys418 cys419 S S• Mixed Acid fermentation Succinate formation PEP carboxylase (heterotrophic CO2 fixation) PEP + CO2 --> oxaloacetate + Pi ATP synthesis by electron transport Formate metabolism Formate: hydrogen lyase Formate --> CO2 + H2 2 enzymes involved, formate dehydrogenase and hydrogenase Note: Enterobacter group also does mixed acid fermentation at neutral pH Mixed Acid Fermentation Glucose Pi Oxaoloacetate malate NADH dehydrogenase PEP carboxylase Pyruvate CoA fumarase H O 2 Fumarate ATP PEP NAD+ Malate ADP CO2 NADH NAD + fumarate reductase Succinate Pyruvate-formate lyase xNAD + xNADH xADP x ATP Glycolysis (See previous notes for complete pathway Not balanced!) ADP ATP Lactate NAD+ NADH Acety-CoA Formate CoA Pi Acetyl-P ADP ATP Acetate Formate-hydrogen lyase + Co + NAD A Acetaldehyde NADH NAD+ Ethanol CO2 H2 Butandiol fermentation Switch to solvent production in low pH New enzymes: Alpha-acetolactate synthase 2 pyruvate --> acetolactate + CO2 TPP as cofactor Butandiol fermentation Reduce acetolactate to acetoin and butandiol. 2,3-Butanediol Fermentation Glucose a -Acetolactate Synthase [ CH 3 C TPP OH PEP ADP ATP Pyruvate CO2 ] CoA Pyruvate OH O CH 3 decarboxylase Acetoin 2,3-Butanediol dehydrogenase C CH 3 COOH a -Acetolactate CO 2 C CH 3 OH O CH C CH 3 OH OH CH CH NADH NAD + 2,3-Butanediol CH 3 CH3 Glycolysis See previous handout for reactions. (not balanced) Lactate Pyruvate-formate lyase Acety-CoA a -Acetolactate xNAD + xNADH xADP xATP CO 2 + Formate NADH CoA + NAD + Acetaldehyde NADH NAD + Ethanol H2 The problem of food and water pollution “..its waters returning, Back to the springs, like the rain, Shall fill them full of refreshment, that which the fountain sends forth returns again to the fountain” All the water on the planet is recycled. Risks of fecal contamination differentiation between fecal and non-fecal enterics is critical Shanks, O. C. et. al. (2006) Competitive Methagenomic DNA Hybridization identifies host-specific microbial genetic markers in cow fecal samples. AEM V 72 N6 p. 4054 – 4060. Simpson, J. M. et. al. (2004). Assessment of equine fecal contamination: the search for alternative bacterial source-tracking targets. FEMS Microbiol. Ecol. V 47 p. 65-75. Dick, L. K., et. al. (2005). Host distributions of uncultivated fecal Bacteriodes bacteria reveal genetic markers for fecal source identification. AEM V71 N6 p. 3184- 3191. Differentiation of Enterics Differentiation based on metabolic characterization Mixed acid Enzyme analysis Intermediate analysis Gas: E. coli, Salmonella No gas: Shigella, S. typhi Butanediol (acetoin) Gas: Enterobacter No gas: Erwinia, Serratia. Summary Free radicals on proteins can also be used to break C-C bonds. Enterics are a good example of reactions. They metabolize pyruvate to most of the products we discussed. ID of enterics critical to assess water quality. Alcohol fermentations Two possibilities: yeast and Zymomonas. Yeast 1815: Gay-Lussac found that yeast made 2 ethanols and 2 carbon dioxides from glucose Buchner: cell-free extract, beginnings of biochemistry Uses glycolytic pathway to make pyruvate Difference from Streptococcus is in what happens to pyruvate New pyruvate enzyme: References: Flores et al. FEMS Micro. Rev. 24: 507-529, 2000; Conway, FEMS Micro. Rev. 103: 1-28, 1992. Summary of the yeast pathway Glucose Glycolytic pathway Net 2 ATP 2 NADH's made 2 pyruvates Oxidative reactions: 3-phosphoglyceraldehyde + Pi + NAD+ -> 1,3-bisphosphoglycerate + NADH Reductive reactions: acetaldehyde + NADH -> ethanol + NAD+ Pyruvate decarboxylase Substrate-level phosphorylation: PEP + ADP -> pyruvate + ATP 2 CO2 1,3-bisphosphoglycerate + ADP -> 3 phosphoglycerate + ATP 2 acetaldehyde H3C C O H Net ATP 2 NADH use 2 ATP make 4 ATP + net of 2 ATP 2 NAD 2 ethanol Pyruvate decarboxylase Pyruvate decarboxylase Pyruvate -> acetaldehyde (CH3CHO) + CO2 Cofactor: thiamine pyrophosphate (TPP). Thus, no oxidation/reduction and no high energy intermediate is made The “active aldehyde” rearranges and forms acetaldehyde as one of the products Function of TPP here is decarboxylation. Zymomonas Natural agent of alcohol fermentations in tropics, isolated from Mexican pulque. Gram negative, motile, small rods, anaerobic to microaerophilic Usually make more than 2 mol ethanol per mol glucose Often more versatile than yeast in substrates used Organism of choice for bulk ethanol production (gasohol) Zymomonas Uses a new pathway for glucose metabolism called Entner-Doudoroff Oxidation of the number one carbon of glucose as in Leuconostoc to form 6phosphogluconate Followed by a dehydration to give a new intermediate: 2-keto-3-deoxy-6phosphogluconate. Glucose ATP ADP Glucose-6-P NADP+ NADPH 6-Phosphogluconate ATP summary used 1 made 2 net = 1 Reoxidation of NAD(P)H H2O 6PG dehydratase 2-Keto-3-deoxy-6-phosphogluconate KD6PG aldolase pyruvate 3-phosphoglyceraldehyde NAD+ Pi NADH 1,3-bisphosphoglycerate ADP ATP 3-phosphoglycerate Pyruvate decarboxylase 2 CO2 2 acetaldehyde Alcohol dehydrogenase pyruvate ATP H2O 2-phosphoglycerate 2 pyruvate ADP phosphoenol pyruvate 2 NAD(P)H 2 NAD(P)+ 2 ethanol Key enzymes and intermediates CO OH HC HO OH CH C HO HC OH HC OH H 2C CO OH H2O O 6-Phosphogluconate dehydratase P O 2-keto-3-deoxy6-phosphoglucon a te (K D6PG) CH HC OH HC OH H 2C O P KD6PG aldolase CO OH HC O C HC OH O CH 3 Pyruvate H 2C O P 3-phosphoglyce raldehyde Summary Pyruvate decarboxylase: uses TPP to decarboxylate pyruvate but only makes acetaldehyde ED pathway: only one G-3-P made, limits ATP production ED pathway oxidizes C-1 of glucose and makes new intermediate, 2-keto-3-deoxy-6-phosphogluconate Extra oxidative reaction in Zymomonas limits ATP production compared to yeast.