Amino Acid BiosynthesisIntroduction

Essential and Non-essential Amino AcidsEssential (10)ArginineHistidineIsoleucineLeucineLysineMethioninePhenylalanineThreonineTryptophanValineNon-essentialAlanineAsparagineAspartateCysteineGlutamateGlutamineGlycineProlineSerineTyrosine

Importance of Balanced DietMilk: proper balance of amino acidsBeans: lysine rich; methionine poorWheat: methionine rich; lysine poor

Bioengineered Wheat: high lysine content

Nitrogen AcquisitionNitrogen FixationNitrate AssimilationAmmonium Assimilation

Nitrogen Fixation(few strains of bacteria)

Nitrifying Bacteria

Nitrate Assimilation(Green plants, some fungi and bacteria)

Ammonium Assimilation (1)(Carbamyl Phosphate Synthetase)

Ammonium Assimilation (2)(Biosynthetic Glutamate Dehydrogenase)and/or(Glutamine Synthetase)

Microbial Nitrogen Acquisition(Metabolic Sources of Organic Nitrogen)

Glutamate (90%)>Amino Acids (90%)

Purines (50%)

Pyrimidines (50%)

Glutamine (10%)>Amino Acids

Amino Sugars

NAD+

PABA

Purines (50%)

Pyrimidines (50%)

Microbial Ammonium Assimilation(High NH3)

Mechanism of Glutamine Synthetase

Microbial Ammonium Assimilation(Low NH3)

Glutamine Synthetase

Glutamate Synthase

(Glutamate Oxoglutarate Aminotransferase, GOGAT)

Glutamine + -ketoglutarate + NADPH > 2 Glutamate + NADP+

Microbial Ammonium AssimilationHigh [NH3]Biosynthetic Glutamate DehydrogenaseGlutamine SynthetaseLow [NH3]Glutamine SynthetaseGlutamate Synthase (Glutamate Oxoglutarate Aminotransferase, GOGAT)

Microbial Ammonium Assimilation(High NH3)

Microbial Ammonium Assimilation(Low NH3)

Role of Glutamate(Nitrogen Donor)

Role of Glutamine(Nitrogen Donor)

Specific Products of GlutamineHistidineTryptophanCarbamyl Phosphate (CPSII)GlucosamineATPCTP

Regulation of Glutamine Synthetase

Pathways of Nitrogen Incorporation

Feedback Inhibition(Nitrogen-sufficient conditions)ProductsHistidineTryptophanCarbamyl-PGlucosamineATPCTP

Nitrogen StatusAlanineSerineGlycin

Modulation of Enzyme Activity

Sensitivity to [NH3]Importance of Glutamine/a-ketoglutarateRatio

Control of Enzyme Activity(Escherichia coli Enzyme)

Control of Gene ExpressionGlnA = Glutamine SynthetaseNtrB = AutokinaseNtrC = Transcription Factor

Control of Gene Expression(NtrC Transcription Factor)

Nitrogen Sufficiency (Rich)(Low Expression of Glutamine Synthesis)Basal expression from P1 (weak promoter)Termination at rho-independent terminatorBasal expression from PinternalRNA polymerase (s70)

Control of Gene Expression(Nitrogen Sufficiency)

Nitrogen Limitation(Ammonia Assimilation = High Expression)Expression from P2 (strong promoter)Requires NtrCP (binds to enhancer)RNA polymerase (s54)Reads through terminatorUpregulation of ntrB and ntrC

Control of Gene Expression(Nitrogen Limitation)

Two Component Sensor-Response Regulator SystemNtrB and NtrC

Two Component Sensor-Response Regulator SystemsAutokinase Sensor Proteins (NtrB) (autophosphorylation of histidine residue)Sensor domainTransmitter domain (C-terminus)Response Regulators (NtrC)N-terminal receiver domainCross-regulation

NtrC

Other NtrC-Sensitive OperonsDirect effect of NtrC-P (e.g. hut operon)Indirect effect of NtrC-P

ConclusionComplex regulation of glutamine synthetase confirms important role in nitrogen metabolism

Biosynthesis of Non-essential Amino AcidsSimple Pathways

Alanine

Aspartate and Asparagine

Glutamate and Glutamine

Proline

Proline (continued)

Arginine(N-Acetylglutamate Synthase)

Arginine (continued)(Acetylglutamate Kinase)

Arginine (continued)(N-Acetyl-g-glutamyl-P Reductase)

Arginine (continued)(N-Acetylornithine g-Aminotransferase)

Arginine (continued)(Acetylornithine Deacetylase)

Arginine (continued)(Acetylornithine:Glutamate Acetyltransferase)

Arginine (continued)(Ornithine Transcarbamylase)

Arginine (continued)(Argininosuccinate Synthetase)

Arginine (continued)(Argininosuccinase)

Arginine Catabolism(Microorganisms)

Regulation of Arginine Metabolism

Arginine Metabolism in Microorganisms(Linear Biosynthetic Pathway)

Arginine Metabolism in Microorganisms(Cyclic Biosynthetic Pathway)

Arginine Metabolism in Microorganisms(Polyamines)

Arginine Metabolism in Microorganisms(Catabolism)

Control of Arginine MetabolismPatterns of Regulation

Arginine Metabolism in Escherichia coli(Properties)Scattered genes (arginine regulon)Linear biosynthetic pathwayAcetylglutamate synthase (AGS)Acetylornithinase (AO)No catabolic pathwaySingle Carbamyl-P synthetase (CPS)Alternative pathway for polyamine synthesisOrnithine Decarboxylase (ODC) orArginine Decarboxylase (ADC) and Agmatine Ureohydrolase (AUH)

Arginine Metabolism in Escherichia coli(Reactions)

Regulation of Enzyme Amount(long-term regulation)Biosynthetic enzymes: repression by arginineCPS: cumulative repression (tandem promoters)ArgininePyrimidinesADC and AUH: induction by arginine

Regulation of Enzyme Activity(short-term regulation)Ornithine synthesis: fbi AGS (no ornithine)CAP synthesis (CPS)Feedback inhibition by UMPActivation by ornithineOTC (no control): no ornithinePolyamines: ADH and AUH

Arginine Metabolism in Bacillus subtilis(Properties)Linear pathwayInducible arginase pathway (no urease)Two CPSs

Arginine Metabolism in B. subtilis(Reactions)

Regulation of Enzyme Amount(long-term regulation)Biosynthetic enzymes: repression by arginineCPSRepression of CPS-A by arginineRepression of CPS-P by pyrimidinesArginase: induction by arginineInducer = arginineNitrogen catabolite repression

Regulation of Enzyme Activity(short-term regulation)Ornithine synthesis: fbi of AGSCAP synthesis (CPS-A and CPS-P)Feedback inhibition of CPS-P by UMPFeedback inhibition of CPS-A by ArgOTC: inhibition by arginase (+arg)Polyamines: Ornithine Decarboxylase (ODC)

Arginine Metabolism in Pseudomonas aeruginosa(Properties)Cyclic pathwayInducible arginase pathway Single CPS

Arginine Metabolism in Pseudomonas aeruginosa(Reactions)

Regulation of Enzyme Amount(long-term regulation)Biosynthetic enzymes: repression by arginineCPS: cumulative repression by arginine and pyrimidinesCatabolic enzymes: induction by arginine

Regulation of Enzyme Activity(short-term regulation)Ornithine synthesis: fbi of AGS and AGKCAP synthesis (CPS)Inhibition by UMPActivation by ornithineOTC: inhibition by argininePolyamines: Ornithine Decarboxylase (ODC)