1. 1/30 6.2 Assimilation of inorganic nitrogen  Many microbes use ammonia (NH 3 ) and nitrate (NO 3 - )as their nitrogen source when organic nitrogen is not available.  Nitrate reductase, nitrite reductase : catalyzing the reduction of nitrate to nitrite and then to ammonia. (1) Dissimilatory nitrate reduction : nitrate is used as an electron acceptor in an anaerobic respiratory process (2) Assimilatory nitrate reduction : utilization of nitrate as a nitrogen source : NAD(P)H is used as the reducing equivalent in many microbes. : Reduced cytochrome provides electrons for nitrate reductase in Pseudomonas (3) Nitrite is further reduced to ammonia by nitrite reductase in a one-step reaction. NADH is the cosubstrate of the enzyme. 6.2.2 Nitrate reduction

2. 2/30 6.2 Assimilation of inorganic nitrogen  Ammonia is assimilated as glutamate by means of two different reactions:  At high ammonia concentrations, glutamate dehydrogenase can assimilate ammonia without consuming ATP since this enzyme has a low affinity for the substrate (Km = 0.1 M).  As low ammonia concentrations, glutamine synthetase with a high substrate affinity (Km = 0.1 mM) assimilates ammonia with consuming ATP.  Glutamate synthase transfers the amino group of glutamine to 2-ketoglutarate. 6.2.3 Ammonia assimilation

3. 3/30 6.2 Assimilation of inorganic nitrogen  Glutamate and glutamine donate amino groups in various synthetic reaction catalyzed by transaminases.  In Escherichia coli, transaminase A, B and C with low specificity synthesize more than ten amino acids.  In addition, glutamate and glutamine are also used as –NH 2 donors in the biosynthetic reactions of various other cell constituents including nucleic acid bases, N-acetyl-glucosamine and the N-acetylmuramic acid of murein.  In E. coli, about 8% of organic nitrogen originates from glutamate and the remaining 15% arises from glutamine. 6.2.3 Ammonia assimilation (continued)

4. 4/30 6.2 Assimilation of inorganic nitrogen  Since glutamine synthetase consumes ATP to assimilate ammonia at low concentrations, its expression as well as its activity is tightly controlled according to ammonia availability to avoid ATP consumption. 6.2.3 Ammonia assimilation (continued)  When glutamine is accumulated, the enzyme is adenylated by adenylyltransferase and then becomes less active than the native form.  It activity is not completely inhibited under ammonia-rich conditions, because glutamine should be synthesized as an amino group donor for the biosynthesis of nucleotides and some amino acids.  Glutamine synthase activity is also controlled by cumulative feedback inhibition (Sec. 12.3.1) by various metabolites synthesized from glutamine.

5. 5/30 6.3 Sulfate assimilation  Sulfur (S) is a constituent of certain amino acids, e.g. methionine and cysteine, as well as various coenzymes.  It also plays an important role in the electron transport chain in Fe-S proteins.  Sulfate (SO 4 2- ) is the major inorganic sulfur source in microbes. (1) Assimilatory sulfate reduction: reduction of sulfate to sulfide (S 2- ) for the biosynthesis of organic sulfur compounds via sulfite (SO 3 2- ). (2) Dissimilatory sulfate reduction: utilization of sulfate as the terminal electron acceptor in a group of anaerobic bacteria known as sulfate-reducing bacteria.

6. 6/30 6.3 Sulfate assimilation  Sulfate is activated by adenosine-5’-phosphosulfate (APS) with ATP, which is further activated to adenosine-3’-phosphate-5’-phosphosulfate (PAPS) (Fig. 6.12).  Cysteine is used as a –SH donor, just as glutamate and glutamine function as –NH 2 donors.  Many bacteria oxidize organic sulfonate, thiols, sulfide or thiophene to sulfate to use as their sulfur source in the absence of sulfate or fulfur-containing amino acids.  These properties have been studied as a means to remove sulfur from petroleum and coal.

7. 7/30 6.4 Amino acid biosynthesis  Amino acids are synthesized using carbon skeletons available from central metabolism, which are pyruvate, oxaloacetate, 2-ketoglutarate, 3-phosphoglycerate, phosphoenolpyruvate, erythrose-4-phosphate and ribose-5-phosphate (Table 6.5)  Some amino acids are synthesized by different pathways depending on the organism.

8. 8/30 6.4 Amino acid biosynthesis  Pyruvate and oxaloacetate are converted to alanine and aspartate by transaminase reaction.  In these reactions, glutamate is the –NH 2 donor.  Asparagine synthetase synthesizes asparagine from aspartate and ammonia consuming ATP, similar to glutamine synthetase reaction. 6.4.1 The pyruvate and oxaloacetate families

9. 9/30 6.4 Amino acid biosynthesis  Threonine, methionine and lysine are produced from aspartate via oxaloacetate in addition to asparagine.  Most prokaryotes employ the diaminopimelate pathway to synthesize lysine. 6.4.1 The pyruvate and oxaloacetate families (continued)

10. 10/30 6.4 Amino acid biosynthesis  Yeasts and fungi synthesize this amino acid from 2-ketoglutarate through the 2-aminoadipate pathway. 6.4.1 The pyruvate and oxaloacetate families (continued)

11. 11/30 6.4 Amino acid biosynthesis  Threonine produced from aspartate is deaminated to 2-ketobutyrate, the precursor for isoleucine biosynthesis.  Two molecules of pyruvate are condensed to 2-acetolactate for production of valine and leucine.  Since pyruvate and 2-ketoburyrate are similar in structure, a series of the same enzymes is used to synthesize isoleucine and valine. 6.4.1 The pyruvate and oxaloacetate families (continued)

12. 12/30 6.4 Amino acid biosynthesis  An EMP pathway intermediate, 3- phosphoglycerate is converted to serine and then further to glycine and cysteine. 6.4.2 The phosphoglycerate family

13. 13/30 6.4 Amino acid biosynthesis  Glutamate synthesized from 2- ketoglutarate by glutamate dehydrogenase is the precursor for the synthesis of proline, arginine and glutamine.  N-acetylornithine deacetylase (rxn 9) has not been detected in coryneform bacteria, P. aeruginosa and S. cerevisiae.  Instead, the reaction is catalyzed by N- acetylglutamate-acetylornithine acetyltransferase, coupling rxn 6 and 9 in these organisms. 6.4.3 The 2-ketoglutarate family

14. 14/30 6.4 Amino acid biosynthesis  The benzene ring of aromatic amino acids is formed from shikimate, which is produced from the condensation of erythrose-4-phosphate and PEP.  Shikimate is further metabolized to chorismate, and then to phenylpyruvate and p-hydroxyphenylpyruvate before being transaminated to phenylalanine and tyrosine, respectively.  Transaminase uses glutamate as the –NH 2 donor. 6.4.4 Aromatic amino acids

15. 15/30 6.4 Amino acid biosynthesis  Tryptophan is synthesized from indole-3-glycerol phosphate catalyzed by tryptophan synthase using serine as the –NH 2 donor.  Indole-3-glycerol phosphate is formed through the condensation of anthranilate and 5-phospho-D-ribosyl-1-phosphate (PRPP).  PRPP synthetase synthesizes PRPP from ribose-5-phosphate and pyrophosphate from ATP.  PRPP is the precursor for the synthesis of histidine and necleotides. 6.4.4 Aromatic amino acids (continued)

16. 16/30 6.4 Amino acid biosynthesis  Histidine is synthesized from PRPP. 6.4.5 Histidine biosynthesis