BRENDA - Enzyme Database show
show all sequences of 6.3.3.6

A conserved tyrosyl-glutamyl catalytic dyad in evolutionarily linked enzymes: carbapenam synthetase and beta-lactam synthetase

Raber, M.L.; Arnett, S.O.; Townsend, C.A.; Biochemistry 48, 4959-40971 (2009)

Data extracted from this reference:

Engineering
Amino acid exchange
Commentary
Organism
E380A
kcat/Km is 74.3fold lower compared to wild-type value
Pectobacterium carotovorum
E380D
retains a functional general base with a pKa of about 7.4 for kcat. The acidic region of the log (kcat/Km) versus pH profiles displays an ionizable group with a pKa of about 7.7. kcat/Km is 1.4fold lower compared to wild-type value
Pectobacterium carotovorum
E380Q
variant displays a plateau in the acidic region. The acidic region of the log (kcat/Km) versus pH profiles becomes pH-independent for E380Q. kcat/Km is 3.8fold lower compared to wild-type value
Pectobacterium carotovorum
Y345A
mutant exhibits a 5fold increase in Km of (2S,5S)-5-carboxymethylproline and a 165fold decrease in specificity constant compared to wild-type enzyme. kcat/Km is 165fold lower compared to wild-type value
Pectobacterium carotovorum
Y345A/E380A
kcat/Km is 87fold lower compared to wild-type value
Pectobacterium carotovorum
Y345F
pH-dependent kcat and kcat/Km plots retain the bell-shaped curve of wild-type CPS with similar pK values. kcat/Km is 1.7fold lower compared to wild-type value
Pectobacterium carotovorum
Y345F/E380D
kcat/Km is 87fold lower compared to wild-type value
Pectobacterium carotovorum
Y345F/E380Q
kcat/Km is 95fold lower compared to wild-type value
Pectobacterium carotovorum
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.075
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380Q
Pectobacterium carotovorum
0.27
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F
Pectobacterium carotovorum
0.35
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, wild-type enzyme
Pectobacterium carotovorum
0.36
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380D
Pectobacterium carotovorum
0.4
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F/E380A
Pectobacterium carotovorum
0.49
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380A; pH 8.0, 25°C, mutant enzyme Y345F/E380D
Pectobacterium carotovorum
0.9
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F/E380Q
Pectobacterium carotovorum
1.7
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345A
Pectobacterium carotovorum
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Pectobacterium carotovorum
-
-
-
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ATP + (2S,5S)-5-carboxymethylproline
catalytic Tyr-Glu dyad is demonstrated by site-directed mutagenesis and kinetic experiments that compare the wild-type enzymes to their respective mutant proteins using pH–rate profiles, 32P-incorporation experiments, solvent isotope effects, proton inventory, and viscosity variation
721613
Pectobacterium carotovorum
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
-
-
?
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
25
-
assay at
Pectobacterium carotovorum
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.0051
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F/E380A
Pectobacterium carotovorum
0.0059
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F/E380D
Pectobacterium carotovorum
0.0067
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380A
Pectobacterium carotovorum
0.011
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345A; pH 8.0, 25°C, mutant enzyme Y345F/E380Q
Pectobacterium carotovorum
0.02
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380Q
Pectobacterium carotovorum
0.17
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F
Pectobacterium carotovorum
0.26
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380D
Pectobacterium carotovorum
0.36
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, wild-type enzyme
Pectobacterium carotovorum
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
E380A
kcat/Km is 74.3fold lower compared to wild-type value
Pectobacterium carotovorum
E380D
retains a functional general base with a pKa of about 7.4 for kcat. The acidic region of the log (kcat/Km) versus pH profiles displays an ionizable group with a pKa of about 7.7. kcat/Km is 1.4fold lower compared to wild-type value
Pectobacterium carotovorum
E380Q
variant displays a plateau in the acidic region. The acidic region of the log (kcat/Km) versus pH profiles becomes pH-independent for E380Q. kcat/Km is 3.8fold lower compared to wild-type value
Pectobacterium carotovorum
Y345A
mutant exhibits a 5fold increase in Km of (2S,5S)-5-carboxymethylproline and a 165fold decrease in specificity constant compared to wild-type enzyme. kcat/Km is 165fold lower compared to wild-type value
Pectobacterium carotovorum
Y345A/E380A
kcat/Km is 87fold lower compared to wild-type value
Pectobacterium carotovorum
Y345F
pH-dependent kcat and kcat/Km plots retain the bell-shaped curve of wild-type CPS with similar pK values. kcat/Km is 1.7fold lower compared to wild-type value
Pectobacterium carotovorum
Y345F/E380D
kcat/Km is 87fold lower compared to wild-type value
Pectobacterium carotovorum
Y345F/E380Q
kcat/Km is 95fold lower compared to wild-type value
Pectobacterium carotovorum
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.075
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380Q
Pectobacterium carotovorum
0.27
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F
Pectobacterium carotovorum
0.35
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, wild-type enzyme
Pectobacterium carotovorum
0.36
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380D
Pectobacterium carotovorum
0.4
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F/E380A
Pectobacterium carotovorum
0.49
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380A; pH 8.0, 25°C, mutant enzyme Y345F/E380D
Pectobacterium carotovorum
0.9
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F/E380Q
Pectobacterium carotovorum
1.7
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345A
Pectobacterium carotovorum
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ATP + (2S,5S)-5-carboxymethylproline
catalytic Tyr-Glu dyad is demonstrated by site-directed mutagenesis and kinetic experiments that compare the wild-type enzymes to their respective mutant proteins using pH–rate profiles, 32P-incorporation experiments, solvent isotope effects, proton inventory, and viscosity variation
721613
Pectobacterium carotovorum
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
-
-
?
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
25
-
assay at
Pectobacterium carotovorum
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.0051
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F/E380A
Pectobacterium carotovorum
0.0059
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F/E380D
Pectobacterium carotovorum
0.0067
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380A
Pectobacterium carotovorum
0.011
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345A; pH 8.0, 25°C, mutant enzyme Y345F/E380Q
Pectobacterium carotovorum
0.02
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380Q
Pectobacterium carotovorum
0.17
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F
Pectobacterium carotovorum
0.26
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380D
Pectobacterium carotovorum
0.36
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, wild-type enzyme
Pectobacterium carotovorum
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.0063
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345A
Pectobacterium carotovorum
0.011
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F/E380Q
Pectobacterium carotovorum
0.012
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F/E380A; pH 8.0, 25°C, mutant enzyme Y345F/E380D
Pectobacterium carotovorum
0.014
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380A
Pectobacterium carotovorum
0.27
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380Q
Pectobacterium carotovorum
0.63
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F
Pectobacterium carotovorum
0.72
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380D
Pectobacterium carotovorum
1.04
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, wild-type enzyme
Pectobacterium carotovorum
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.0063
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345A
Pectobacterium carotovorum
0.011
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F/E380Q
Pectobacterium carotovorum
0.012
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F/E380A; pH 8.0, 25°C, mutant enzyme Y345F/E380D
Pectobacterium carotovorum
0.014
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380A
Pectobacterium carotovorum
0.27
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380Q
Pectobacterium carotovorum
0.63
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme Y345F
Pectobacterium carotovorum
0.72
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, mutant enzyme E380D
Pectobacterium carotovorum
1.04
-
(2S,5S)-5-carboxymethylproline
pH 8.0, 25°C, wild-type enzyme
Pectobacterium carotovorum
Other publictions for EC 6.3.3.6
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
743905
Hamed
-
Stereoselective production of ...
Pectobacterium carotovorum
ACS Catal.
7
1279-1285
2017
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1
1
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1
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5
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1
1
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5
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727703
Tahlan
Origins of the beta-lactam rin ...
Pectobacterium carotovorum, Streptomyces cattleya
J. Antibiot.
66
401-410
2013
-
-
-
-
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-
-
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2
-
3
-
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2
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2
-
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2
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2
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2
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721613
Raber
A conserved tyrosyl-glutamyl c ...
Pectobacterium carotovorum
Biochemistry
48
4959-40971
2009
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-
-
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8
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8
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1
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1
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1
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8
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8
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8
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1
-
1
-
-
8
-
-
-
-
-
-
-
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8
8
721605
Arnett
Rate-limiting steps and role o ...
Pectobacterium carotovorum
Biochemistry
46
9337-9345
2007
-
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1
-
2
-
-
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1
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1
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1
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1
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1
1
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1
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2
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1
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1
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1
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1
1
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1
1
721590
Gerratana
Inhibition and alternate subst ...
Pectobacterium carotovorum
Biochemistry
42
7836-7847
2003
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1
-
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4
4
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2
1
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4
-
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1
-
-
-
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4
1
1
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4
1
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9
-
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1
-
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4
9
4
-
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2
1
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1
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4
1
1
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4
1
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1
1
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4
4
722636
Miller
Crystal structure of carbapena ...
Pectobacterium carotovorum
J. Biol. Chem.
278
40996-41002
2003
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1
1
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1
2
1
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1
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1
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1
1
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1
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1
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1
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1
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1
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1
1
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