An introduction the Amino Acid Biosynthesis.ppt

The Amino Acid Biosynthesis

1. Amino Acid Biosynthesis
Introduction

2. Essential and Non-essential
Amino Acids
•Essential (10)
–Arginine
–Histidine
–Isoleucine
–Leucine
–Lysine
–Methionine
–Phenylalanine
–Threonine
–Tryptophan
–Valine
•Non-essential
–Alanine
–Asparagine
–Aspartate
–Cysteine
–Glutamate
–Glutamine
–Glycine
–Proline
–Serine
–Tyrosine

3. Importance of Balanced Diet
•Milk: proper balance of amino acids
•Beans: lysine rich; methionine poor
•Wheat: methionine rich; lysine poor
•Bioengineered Wheat: high lysine
content

4. Nitrogen Acquisition
•Nitrogen Fixation
•Nitrate Assimilation
•Ammonium Assimilation

5. Nitrogen Fixation
(few strains of bacteria)
N2 + 8H
+
+ 8e
–
+ 16ATP + 16 H2O
2NH
3 + H
2 + 16ADP + 16P
i
Nitrogenase

6. Nitrifying Bacteria
N H
3
N O
3
–
"O xidation"

7. Nitrate Assimilation
(Green plants, some fungi and bacteria)
NO
3
–
+ NADH + H
+
NO
2
–
+ H
2
O + NAD
+
Nitrate Reductase
NO
2
–
+ 8H
+
+ 6e
–
NH
4
+
+ 2H
2
O
Nitrite Reductase

8. Ammonium Assimilation (1)
(Carbamyl Phosphate Synthetase)
H
2
N C
O
O P
2A T P 2A D P + P
i
N H
3
+ H C O
3
–

9. Ammonium Assimilation (2)
(Biosynthetic Glutamate Dehydrogenase)
and/or
(Glutamine Synthetase)
N H
3
G lutam ate
N H
3
G lutam ine

10. Microbial Nitrogen Acquisition
(Metabolic Sources of Organic Nitrogen)

11. Microbial Ammonium Assimilation
(High NH
3
)
+
H
3
N C COO
–
CH
2
CH
2
COO
–
H
Glutamate
NH
3
+ NADPH NADP
+
"Biosynthetic"
Glutamate Dehydrogenase
O
C COO
–
CH
2
CH
2
COO
–
α -Ketoglutarate
+
H
3
N C COO
–
CH
2
CH
2
COO
–
H
Glutamate
ATP + NH
3
ADP + P
i
Glutamine Synthetase
+
H
3
N C COO
–
CH
2
CH
2
C
H
O NH
2
Glutamine

12. Mechanism of Glutamine Synthetase
+
H
3
N C COO
–
CH
2
CH
2
COO
–
H
Glutamate
ATP ADP
Glutamine
Synthetase
+
H
3
N C COO
–
CH
2
CH
2
COO P
H
NH
3
P
i
Glutamine
Synthetase
+
H
3
N C COO
–
CH
2
CH
2
C
H
O NH
2
Glutamine
γ-Glutamyl-P

13. Microbial Ammonium Assimilation
(Low NH
3
)
Glutamine Synthetase
Glutamate Synthase
(Glutamate Oxoglutarate Aminotransferase, GOGAT)
Glutamine + α-ketoglutarate + NADPH > 2——— Glutamate + NADP
+

14. Microbial Ammonium Assimilation
•High [NH
3]
–“Biosynthetic” Glutamate Dehydrogenase
–Glutamine Synthetase
•Low [NH
3]
–Glutamine Synthetase
–Glutamate Synthase (Glutamate Oxoglutarate
Aminotransferase, GOGAT)

15. Microbial Ammonium Assimilation
(High NH
3
)
OCCOO
–
CH
2
CH
2
COO
–
+
H
3NCCOO
–
CH
2
CH
2
COO
–
H
NH
3 + NADPH NADP
+
"Biosynthetic"
Glutamate Dehydrogenase
α-Ketoglutarate Glutamate
+
H
3NCCOO
–
CH
2
CH
2
COO
–
H
+
H
3NCCOO
–
CH
2
CH
2
C
H
O NH
2
ATP + NH
3 ADP + P
i
Glutamine Synthetase
Glutamate Glutamine

16. Microbial Ammonium Assimilation
(Low NH
3
)
+
H
3NCCOO
–
CH
2
CH
2
COO
–
H
+
H
3NCCOO
–
CH
2
CH
2
C
H
O NH
2
ATP + NH
3
ADP + P
i
Glutamine Synthetase
Glutamate Glutamine
+
H
3NCCOO
–
CH
2
CH
2
C
H
O NH
2
OCCOO
–
CH
2
CH
2
COO
–
NADPH NADP
+
+
H
3NCCOO
–
CH
2
CH
2
COO
–
H
Glutamine α-Ketoglutarate Glutamate
+
Glutamate Synthase

17. Role of Glutamate
(Nitrogen Donor)
Glu αKG
R RNH
2

18. Role of Glutamine
(Nitrogen Donor)
Gln Glu
R RNH
2

19. Specific Products of Glutamine
•Histidine
•Tryptophan
•Carbamyl Phosphate (CPSII)
•Glucosamine
•ATP
•CTP

20. Regulation of Glutamine
Synthetase

21. Pathways of Nitrogen
Incorporation
Glu Gln
"Nitrogen"
CompoundsGS

22. Feedback Inhibition
(Nitrogen-sufficient conditions)
•Products
–Histidine
–Tryptophan
–Carbamyl-P
–Glucosamine
–ATP
–CTP
•Nitrogen Status
–Alanine
–Serine
–Glycin

23. Modulation of Enzyme Activity
Sensitivity to [NH
3]
Importance of
Glutamine/-ketoglutarate
Ratio

24. Control of Enzyme Activity
(Escherichia coli Enzyme)
P
II OH
Adenylyltransferase
•
P
II OUMP
Adenylyltransferase
•
UTP
PP
i
UMP
H
2
O
ATP PP
i
Glutamine Synthetase
OH
Glutamine Synthetase
OAMP
(More Active)
No Feedback Inhibition
(Less Active)
Sensitive to fbi
Uridylyl
Transferase
Uridylyl
Removing
Enzyme
ADP P
i
Nitrogen-limited
Growth Conditions
Nitrogen-Rich
Growth Conditions
Role = NH
3 Assimilation
and Anabolism
Role = Glutamine Synthesis
for Anabolism
(+) α-KGGlutamine (+)

25. Control of Gene Expression
P
1
P
2
glnA ntrBntrC
P
internal
rho-independent terminator
GlnA = Glutamine Synthetase
NtrB = Autokinase
NtrC = Transcription Factor

26. Control of Gene Expression
(NtrC Transcription Factor)
P
II OH
NtrC–P NtrC
UTP
PP
i
UMP
H
2
O
P
II OUMP
ATP ADP
(Active)
(Inactive)
Uridylyl
Transferase
Uridylyl
Removing
Enzyme
H
2
O P
i
Nitrogen-limited
Growth Conditions
Nitrogen-Rich
Growth Conditions
Transcription from P
2
using
RNA Polymerase (σ
54
)
Anabolism
(+) α-KG
Glutamine (+)
NtrB
°
NtrB-P
Basal Transcription from P
1
using
RNA Polymerase (σ
70
)
Anabolism
NH
3
Assimilation
NtrB
Autophosphorylation

27. Nitrogen Sufficiency (Rich)
(Low Expression of Glutamine Synthesis)
•Basal expression from P
1
(weak promoter)
•Termination at rho-independent terminator
•Basal expression from P
internal
•RNA polymerase (
70
)

28. Control of Gene Expression
(Nitrogen Sufficiency)
P
1
P
2
glnA ntrBntrC
P
internal
rho-independent terminator
mRNAs

29. Nitrogen Limitation
(Ammonia Assimilation = High Expression)
•Expression from P
2 (strong promoter)
–Requires NtrC–P (binds to enhancer)
–RNA polymerase (
54
)
•Reads through terminator
–Upregulation of ntrB and ntrC

30. Control of Gene Expression
(Nitrogen Limitation)
P
1
P
2
glnA ntrBntrC
P
internal
rho-independent terminator
mRNA

31. Two Component Sensor-Response
Regulator System
NtrB and NtrC

32. Two Component Sensor-Response
Regulator Systems
•Autokinase Sensor Proteins (NtrB)
(autophosphorylation of histidine residue)
–Sensor domain
–Transmitter domain (C-terminus)
•Response Regulators (NtrC)
–N-terminal receiver domain
–Cross-regulation

33. NtrC
ADP + P
i
Receiver
Domain
Transcriptional
Activation
DNA
Binding
ATP
Binding Motif
ATP

34. Other NtrC-Sensitive Operons
•Direct effect of NtrC-P
(e.g. hut operon)
•Indirect effect of NtrC-P

35. Conclusion
Complex regulation of
glutamine synthetase confirms
important role in nitrogen
metabolism

36. Biosynthesis of Non-essential
Amino Acids
Simple Pathways

37. Alanine
+
H
3
N C
H
CH
3
COO
–
H
3
C C COO
–
O
Alanine
Pyruvate
Amino
Acid
α -Keto
Acid
Aminotransferase

38. Aspartate and Asparagine
+
H
3
N C COO
–
CH
2
COO
–
H
Aspartate
O
C COO
–
CH
2
COO
–
Oxaloacetate
Amino
Acid
α -Keto
Acid
Aminotransferase
Asparagine
Synthetase
Gln
+
ATP
Glu
+
AMP + PP
i
+
H
3
N C COO
–
CH
2
C
H
O NH
2
Asparagine

39. Glutamate and Glutamine
OCCOO
–
CH
2
CH
2
COO
–
+
H
3
NCCOO
–
CH
2
CH
2
COO
–
H
+
H
3NCCOO
–
CH
2
CH
2
C
H
O NH
2Amino
Acid
α-Keto
Acid
α-Ketoglutarate Glutamate
Aminotransferase
ATP + NH
3 ADP + P
i
Glutamine Synthetase
Glutamine

40. Proline
+
H
3
N C COO
–
CH
2
CH
2
COO
–
H
Glutamate
ATP ADP
+
H
3
N C COO
–
CH
2
CH
2
COO P
H
+
H
3
N C COO
–
CH
2
CH
2
C
H
H O
Glutamate-5-P Glutamate--5-
Semialdehyde
γ-Glutamyl
Kinase
γ-Glutamyl-P
Reductase
NAD(P)H
+
H
+
NAD(P)
+
+
P
i

41. Proline (continued)
+
H
3
N C COO
–
CH
2
CH
2
C
H
H O
Glutamate--5-
Semialdehyde
Nonenzymatic
N
H
COO
–
Pyrroline-5-Carboxylate
Reductase
N
COO
–
H
2
Pyrroline-5-Carboxylate
Proline
NAD(P)H + H
+
NAD(P)
+
H
2
O

42. Arginine
(N-Acetylglutamate Synthase)
+
H
3
N C COO
–
CH
2
CH
2
COO
–
H
Glutamate
Acetyl–CoA CoASH
N-Acetylglutamate
Synthase
N
H
C COO
–
CH
2
CH
2
COO
–
H
C
O
H
3
C
N -Acetylglutamate

43. Arginine (continued)
(Acetylglutamate Kinase)
ATP ADP
N
H
C COO
–
CH
2
CH
2
COO
–
H
C
O
H
3
C
N -Acetylglutamate
Acetylglutamate
Kinase
N
H
C COO
–
CH
2
CH
2
C
H
C
O
H
3
C
O O P
O
O
–
O
–
N -Acetylglutamate-5-P

44. Arginine (continued)
(N-Acetyl--glutamyl-P Reductase)
N
H
C COO
–
CH
2
CH
2
COO P
H
C
O
H
3
C
N -Acetylglutamate-5-P
NAD(P)
+
+
P
i
N-Acetyl- γ-glutamyl P
Reductase
N
H
C COO
–
CH
2
CH
2
C
H
C
O
H
3
C
H O
N -Acetylglutamate-5-
Semialdehyde
NAD(P)H
+
H
+

45. Arginine (continued)
(N-Acetylornithine -Aminotransferase)
N
H
C COO
–
CH
2
CH
2
C
H
C
O
H
3
C
H O
N -Acetylglutamate-5-
Semialdehyde
Glu α KG
N-Acetylornithine
γ-Aminotransferase
N
H
C COO
–
CH
2
CH
2
CH
2
H
C
O
H
3
C
NH
3
+
N -Acetylornithine

46. Arginine (continued)
(Acetylornithine Deacetylase)
N
H
C COO
–
CH
2
CH
2
CH
2
H
C
O
H
3
C
NH
3
+
N -Acetylornithine
Acetylornithine
Deacetylase
H
2
O CH
3
COO
–
+
H
3
N C COO
–
CH
2
CH
2
CH
2
H
NH
3
+
Ornithine

47. Arginine (continued)
(Acetylornithine:Glutamate Acetyltransferase)
N
H
C COO
–
CH
2
CH
2
CH
2
H
C
O
H
3
C
NH
3
+
N -Acetylornithine
Acetylornithine:
Glutamate
Acetyltransferase
Glu Acetylglutamate
+
H
3
N C COO
–
CH
2
CH
2
CH
2
H
NH
3
+
Ornithine

48. Arginine (continued)
(Ornithine Transcarbamylase)
H
2
NC
O
OP
+
H
3
NCCOO
–
H
(CH
2
)
3
NH
3
+
+
H
3
NCCOO
–
H
(CH
2
)
3
NH
C
ONH
2
Ornithine
+
P
i
Citrulline
Carbamyl P
Non-standard amino acids
(not present in proteins)

49. Arginine (continued)
(Argininosuccinate Synthetase)
+
H
3
N C COO
–
H
(CH
2
)
3
NH
C
O NH
2
Citrulline
+ C
+
H
3
N COO
–
H
H
2
C COO
–
Aspartate
ATP AMP + PP
i
+
H
3
N C COO
–
H
(CH
2
)
3
NH
C
H
2
N
H
N C
H
COO
–
H
2
C COO
–
Argininosuccinate

50. Arginine (continued)
(Argininosuccinase)
+
H
3
N C COO
–
H
(CH
2
)
3
NH
C
H
2
N
H
N C
H
COO
–
H
2
C COO
–
Argininosuccinate
+
H
3
N C COO
–
H
(CH
2
)
3
NH
C
H
2
N
NH
2
Arginine
Fumarate
C C
COO
–
H
H
–
OOC

51. Arginine Catabolism
(Microorganisms)
+
H
3NCCOO
–
H
(CH
2)
3
NH
C
H
2
N NH
2
+
H
3NCCOO
–
H
(CH
2)
3
NH
3
+
+
H
3NCCOO
–
H
(CH
2)
2
CHO
+
H
3NCCOO
–
H
(CH
2)
2
COO
–
H
2
O Urea
Arginase
Ornithine
Arginine
aKG Glu
Ornithine-δ-
Aminotransferase
Glutamate-5-
Semialdehyde
NAD(P)
+
+ H
2
O
NAD(P)H + H
+
Glutamate
Glutamate
Semialdehyde
Dehydrogenase

52. Regulation of Arginine
Metabolism

53. Arginine Metabolism in Microorganisms
(Linear Biosynthetic Pathway)
AcGSA AcOrn Orn Cit
CAP
CPS
OTC
AcGluPAcGlu
OTC
Glu
AcCoACoA
ASA Arg
Biosynthetic Steps
Enzymes
Arginine
AGK

54. Arginine Metabolism in Microorganisms
(Cyclic Biosynthetic Pathway)
AcGSA AcOrn Orn Cit ASA Arg
CAP
CPS
OTC
AcGluP
OTC
AcGluGlu
AGS
AGK
Biosynthetic Steps
Enzymes
Arginine
AOGAT

55. Arginine Metabolism in Microorganisms
(Polyamines)
ADCAUH
Cit ASA Arg
OTC
ODC
Orn
CAP
CPS
AcOrnAcGSAAcGluP
OTC
AcGluGlu
AGS
AGK
Biosynthetic Steps
Enzymes
Arginine
AOGAT
Polyamines

56. Arginine Metabolism in Microorganisms
(Catabolism)
ADCAUH
Cit ASA Arg
OTC
AGA
ODC
Orn
CAP
CPS
AcOrnAcGSA
P5CA
AcGluP
OTC
AcGluGlu
AGS AGK
Biosynthetic Steps
Enzymes
Arginine
AOGAT
PolyaminesGSA
Catabolic Steps
OTAP5CADH

57. Control of Arginine
Metabolism
Patterns of Regulation

58. Arginine Metabolism in Escherichia coli
(Properties)
•Scattered genes (arginine regulon)
•Linear biosynthetic pathway
–Acetylglutamate synthase (AGS)
–Acetylornithinase (AO)
•No catabolic pathway
•Single Carbamyl-P synthetase (CPS)
•Alternative pathway for polyamine synthesis
–Ornithine Decarboxylase (ODC) or
–Arginine Decarboxylase (ADC) and Agmatine Ureohydrolase (AUH)

59. Arginine Metabolism in Escherichia coli
(Reactions)
ODC
AcGSA AcOrn Orn Cit
CAP
CPS
OTC
Asp
ATC
AcGluPAcGlu
OTC
Glu
AcCoACoA
H
2
OAcOH
ASA
UMP (UTP, CTP)
AUH
Arg
ADC
Polyamines
AGS AGK
Pyrimidines
Biosynthetic Steps
Catabolic Steps
Enzymes
Arginine
AO

60. Regulation of Enzyme Amount
(long-term regulation)
•Biosynthetic enzymes: repression by
arginine
•CPS: cumulative repression (tandem
promoters)
–Arginine
–Pyrimidines
•ADC and AUH: induction by arginine

61. Regulation of Enzyme Activity
(short-term regulation)
•Ornithine synthesis: fbi AGS
(no ornithine)
•CAP synthesis (CPS)
–Feedback inhibition by UMP
–Activation by ornithine
•OTC (no control): no ornithine
•Polyamines: ADH and AUH

62. Arginine Metabolism in Bacillus subtilis
(Properties)
•Linear pathway
•Inducible “arginase” pathway
(no urease)
•Two CPS’s

63. Arginine Metabolism in B. subtilis
(Reactions)
ODC
AcGSA AcOrn Orn Cit ASA Arg
CAP
CPS-A
CPS-P
OTC
Asp UMP (UTP, CTP)
ATC
AcGluPAcGlu
OTC
Glu
Polyamines
AGA
AGS AGK
Pyrimidines
GSA
Biosynthetic Steps
Catabolic Steps
Enzymes
Arginine

64. Regulation of Enzyme Amount
(long-term regulation)
•Biosynthetic enzymes: repression by
arginine
•CPS
–Repression of CPS-A by arginine
–Repression of CPS-P by pyrimidines
•Arginase: induction by arginine
–Inducer = arginine
–Nitrogen catabolite repression

65. Regulation of Enzyme Activity
(short-term regulation)
•Ornithine synthesis: fbi of AGS
•CAP synthesis (CPS-A and CPS-P)
–Feedback inhibition of CPS-P by UMP
–Feedback inhibition of CPS-A by Arg
•OTC: inhibition by arginase (+arg)
•Polyamines: Ornithine Decarboxylase
(ODC)

66. Arginine Metabolism in Pseudomonas aeruginosa
(Properties)
•Cyclic pathway
•Inducible “arginase” pathway
•Single CPS

67. Arginine Metabolism in Pseudomonas aeruginosa
(Reactions)
ODC
AcGSA AcOrn Orn Cit ASA Arg
CAP
CPS
OTC
Asp UMP (UTP, CTP)
ATC
AcGluP
OTC
AcGluGlu
Polyamines
AGA
AGS AGK
Pyrimidines
GSA
Biosynthetic Steps
Catabolic Steps
Enzymes
Arginine

68. Regulation of Enzyme Amount
(long-term regulation)
•Biosynthetic enzymes: repression by
arginine
•CPS: cumulative repression by arginine
and pyrimidines
•Catabolic enzymes: induction by
arginine

69. Regulation of Enzyme Activity
(short-term regulation)
•Ornithine synthesis: fbi of AGS and AGK
•CAP synthesis (CPS)
–Inhibition by UMP
–Activation by ornithine
•OTC: inhibition by arginine
•Polyamines: Ornithine Decarboxylase (ODC)