Download 6.2 Assimilation of inorganic nitrogen

발효화학 (Fermentation Chemistry), Bacterial Physiology and Metabolism
Chapter 6. Biosynthesis and
microbial growth
Yong-Cheol Park
(http://park.openwetware.org)
Department of Advanced Fermentation
Fusion Science & Technology
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6.1 Molecular composition of bacterial cells
 The elements in the cells
make up a range of molecules
with various functions.
 Cellular molecular
composition varies depending
on the strain and growth
conditions.
 When Escherichia coli grows
in a glucose-mineral salts
medium, its molecular
composition during the
logarithmic phase is presented
in Table 6.1 and its water
content is over 70%.
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6.1 Molecular composition of bacterial cells
 The catabolism supplies carbon skeletons for monomer synthesis followed by
their polymerization and assembly into cell structure.
 For anabolism, nitrogen (N), sulfur (S), phosphorus (P) and so on are needed in
addition to the carbon skeletons.
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6.2 Assimilation of inorganic nitrogen
 Many cell constituents contains nitrogen
such as amino acids and nucleic acid
bases.
 Organic nitrogen is used preferentially
over inorganic nitrogen by almost all
microbes.
 Nitrogen fixation : Reduction of gaseous
nitrogen to ammonia to meet the
nitrogen requirement in some
prokaryotes when organic nitrogen such
as ammonia or nitrate is not available.
 Nitrogen fixation is not found in
eukaryotes.
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6.2 Assimilation of inorganic nitrogen
6.2.1 Nitrogen fixation
 Nitrogen is incorporated into cell constituents through transamination
reactions using glutamate or glutamine as the amino group donor.
 Glutamate and glutamine are synthesized from ammonia.
 Gaseous nitrogen (N2) is very stable
as it possesses a triple covalent bond.
 When organic nitrogen (fixed nitrogen)
is not available, some prokaryotes
reduce the stable N2 to ammonia by
using a large amount of ATP and
reduced electron carriers.
 The fixed nitrogen returns to gaseous
nitrogen though nitrification (Sec. 10.2)
and denitrification (Sec. 9.1)
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6.2 Assimilation of inorganic nitrogen
6.2.1.1 Biochemistry of N2 fixation
 Nitrogen reduction to ammonia :
 This is an exergonic reaction, but require a high activation energy due to the
stable triple bond in N2.
 Nitrogen fixing microbes produce ‘nitrogenase’ reducing nitrogen under
normal physiological conditions.
6.2.1.1.1 Nitrogenase
 Nitrogenase is a complex protein
composed of azoferredoxin and
molybdeferredoxin in a 2:1 ratio.
 Both the enzymes are [Fe-S]
proteins, and molybdoferredoxin
contains molybdenum.
 Molybdoferredoxin reduces nitrogen
with the reducing equvalents
provided by azoferredoxin.
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6.2 Assimilation of inorganic nitrogen
6.2.1.1.1 Nitrogenase (continued)
 Molybdoferredoxin binds N2
before forming a nitrogenase
complex with the ATP-reduced
azoferredoxin complex.
 At this points, electrons are
transferred from azoferredoxin
to molybdoferredozin with ATP
hydrolysis.
 Since azoferredoxin is a one
electron carrier, the reduction
of a nitrogen molecule
requires six oxidationreduction cycles with the
hydrolysis of at least 16 ATP
molecules.
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6.2 Assimilation of inorganic nitrogen
6.2.1.1.1 Nitrogenase (continued)
 Nitrogenase can reduce various other substance in addition to dinitrogen.
6.2.1.1.2 Electron carriers
 Ferredoxin or flavodoxin supply the electrons required for nitrogen reduction.
 Photosynthetic organisms reduce ferredoxin by light reactions.
 Obilgate anaerobes reduce ferredoxin by pyruvate:ferredoxin oxidoreductase or
hydrogenase.
 Aerobes reduce them through a reverse electron transport mechanism using
reduced pyridine nucleotides (Section 10.1).
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6.2 Assimilation of inorganic nitrogen
6.2.1.5 Regulation of N2 fixation
 To avoid the waste of energy, N2 fixation is regulated both at the transcriptional
level and by controlling enzyme activity.
 Nitrogenase activity is inhibited by ammonia and under starvation conditions
with a low EC.
 Ammonia switch – quick and reversible inhibition by ammonia
: Ammonia accumulation An arginine residue of azoferredoxin is bound with
ADP-ribose from NAD+.  The nitrogenase complex is inactive with ADP-ribose.
 Ammonia inhibits the transcription of genes of nitrogenase complex (nif regulon
consisting of 7 operons with 20 gene in Klebsiella pneumoniae (Fig. 6.8)).
 In addition to these genes, nitrogen control genes, ntr, are also involved in their
regulation.
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