Cloned (Comment) | Organism |
---|---|
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain HB101 | Pseudomonas putida |
Crystallization (Comment) | Organism |
---|---|
purified recombinant wild-type and mutant C116H enzymes, the latter complexed with L-methionine or L-homocysteine, sitting drop vapour diffusion method, mixing of 500 nl of 20 mg/ml protein 10 mM HEPES-NaOH, pH 7.4, with 500 nl of reservoir solution containing 200 mM MES-NaOH, pH 6.2, with 12.5% w/v PEG6000, 250 mm ammonium sulfate, 0.5 mM pyridoxal 5'-phosphate, and 0.5% v/v 2-mercaptoethanol, and equilibration against 0.1 ml of reservoir solution, 20°C, for ligand binding soaking of crystals in reservoir solution containing 50 mM amino acid, method optimization, X-ray diffraction structure determination and analysis at 2.1-2.6 A resolution, molecular replacement | Pseudomonas putida |
Protein Variants | Comment | Organism |
---|---|---|
C116H | site-directed mutagenesis, the H-bond network of the mutant is broken affecting catalytic residue Tyr114, the mutant acquires a beta-elimination activity, and shows high conformational changes upon substrate binding, implicating an altered substrate specificity, highly increased for L-cysteine and reduced for L-methionine, compared to the wild-type enzyme | Pseudomonas putida |
C116H/Y114F | site-directed mutagenesis, almost inactive mutant in gamma- and beta-elimination reactions of amino acids with 2-mercaptoethanol, no activity with L-methionine | Pseudomonas putida |
D241E | site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme | Pseudomonas putida |
D241H | site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme | Pseudomonas putida |
D241I | site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme | Pseudomonas putida |
D241K | site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme | Pseudomonas putida |
D241M | site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme | Pseudomonas putida |
D241N | site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme | Pseudomonas putida |
D241R | site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme | Pseudomonas putida |
D241T | site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme | Pseudomonas putida |
K240D | site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme | Pseudomonas putida |
K240E | site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme | Pseudomonas putida |
K240H | site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme | Pseudomonas putida |
K240M | site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme | Pseudomonas putida |
K240R | site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme | Pseudomonas putida |
K240S | site-directed mutagenesis | Pseudomonas putida |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
DL-propargylglycine | inhibition profiles of wild-type and mutant enzymes, overview | Pseudomonas putida |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.04 | - |
L-methionine | pH 8.0, 37°C, recombinant mutant K240R | Pseudomonas putida | |
0.2 | - |
L-cysteine | pH 8.0, 37°C, recombinant wild-type enzyme | Pseudomonas putida | |
0.2 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutant Y114F/C116H | Pseudomonas putida | |
0.2 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant Y114F/C116H | Pseudomonas putida | |
0.3 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutant C166H | Pseudomonas putida | |
0.3 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutants K240R and K240M | Pseudomonas putida | |
0.4 | - |
S-methyl-L-cysteine | pH 8.0, 37°C, recombinant mutant Y114F/C116H | Pseudomonas putida | |
0.5 | - |
L-methionine | pH 8.0, 37°C, recombinant wild-type enzyme | Pseudomonas putida | |
0.6 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant K240R | Pseudomonas putida | |
0.7 | - |
S-methyl-L-cysteine | pH 8.0, 37°C, recombinant wild-type enzyme | Pseudomonas putida | |
0.8 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant K240N | Pseudomonas putida | |
1 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutants K240I and K240N | Pseudomonas putida | |
1.1 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant wild-type enzyme | Pseudomonas putida | |
1.4 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant C166H | Pseudomonas putida | |
1.4 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant K240M | Pseudomonas putida | |
2.1 | - |
S-methyl-L-cysteine | pH 8.0, 37°C, recombinant mutant C166H | Pseudomonas putida | |
2.3 | - |
L-methionine | pH 8.0, 37°C, recombinant mutant C166H | Pseudomonas putida | |
4.8 | - |
L-methionine | pH 8.0, 37°C, recombinant mutant K240I | Pseudomonas putida | |
5.7 | - |
L-methionine | pH 8.0, 37°C, recombinant mutant K240M | Pseudomonas putida | |
6 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant K240I | Pseudomonas putida | |
9.8 | - |
L-methionine | pH 8.0, 37°C, recombinant mutant K240N | Pseudomonas putida |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas putida | P13254 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from Escherichia coli strain HB101 by PEG 6000 fractionation, dialysis, folowed by anion exchange chromatography, and gel filtration | Pseudomonas putida |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
DL-homocysteine + 2-mercaptoethanol | gamma-replacement reaction, wild-type enzyme | Pseudomonas putida | H2S + S-(beta-hydroxyethyl)-L-homocysteine | - |
? | |
L-cysteine + 2-mercaptoethanol | beta-replacement reaction, wild-type enzyme and mutant C116H | Pseudomonas putida | H2S + S-(beta-hydroxyethyl)-L-cysteine | - |
? | |
L-methionine + 2-mercaptoethanol | gamma-replacement reaction, wild-type enzyme | Pseudomonas putida | methanethiol + S-(beta-hydroxyethyl)-L-homocysteine | - |
? | |
S-methyl-L-cysteine + 2-mercaptoethanol | - |
Pseudomonas putida | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
L-methionine gamma-lyase | - |
Pseudomonas putida |
MGL | - |
Pseudomonas putida |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Pseudomonas putida |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
50 | - |
purified wild-type enzyme and mutants, all completely stable, except for mutants D241H and D241I loosing about 10% activity | Pseudomonas putida |
70 | - |
purified mutant D241R, loss of 70% activity | Pseudomonas putida |
75 | - |
purified wild-type enzyme and mutants D241T, D241M, D241N, D241E, D241H, D241K, K240S, K240R, and K240C completely stable up to, loss of 35% activity for mutant K240N, of 15% for D241I, of 85% for mutant K240M, and of 95% for mutant K240I and D241R, profiles, overview | Pseudomonas putida |
80 | - |
purified wild-type enzyme and mutants, inactivation of all besides mutants D241E, and K240N, retaining 20% and K240M retaining 10% activity, respectively | Pseudomonas putida |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.03 | - |
L-methionine | pH 8.0, 37°C, recombinant mutant C166H | Pseudomonas putida | |
0.12 | - |
S-methyl-L-cysteine | pH 8.0, 37°C, recombinant mutant Y114F/C116H | Pseudomonas putida | |
0.89 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutant Y114F/C116H | Pseudomonas putida | |
1.06 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutant K240N | Pseudomonas putida | |
1.36 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant Y114F/C116H | Pseudomonas putida | |
1.45 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutant K240R | Pseudomonas putida | |
1.58 | - |
S-methyl-L-cysteine | pH 8.0, 37°C, recombinant wild-type enzyme | Pseudomonas putida | |
1.66 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutant K240M | Pseudomonas putida | |
2.13 | - |
L-cysteine | pH 8.0, 37°C, recombinant wild-type enzyme | Pseudomonas putida | |
2.23 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutant K240I | Pseudomonas putida | |
5.03 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant K240N | Pseudomonas putida | |
22.7 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant K240R | Pseudomonas putida | |
23.3 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutant C166H | Pseudomonas putida | |
28.8 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant K240M | Pseudomonas putida | |
33.4 | - |
L-methionine | pH 8.0, 37°C, recombinant wild-type enzyme | Pseudomonas putida | |
46.3 | - |
S-methyl-L-cysteine | pH 8.0, 37°C, recombinant mutant C166H | Pseudomonas putida | |
48.4 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant C166H | Pseudomonas putida | |
62.2 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant K240I | Pseudomonas putida | |
71 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant wild-type enzyme | Pseudomonas putida |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Pseudomonas putida |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
pyridoxal 5'-phosphate | - |
Pseudomonas putida |
General Information | Comment | Organism |
---|---|---|
additional information | general acid catalytic residue is conserved Tyr114, conserved residue Lys240 is important for substrate recognition and structural stability, conserved residue Asp241 is also important in the elimination reaction, structure-function analysis, overview | Pseudomonas putida |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.01 | - |
L-methionine | pH 8.0, 37°C, recombinant mutant C166H | Pseudomonas putida | |
0.3 | - |
S-methyl-L-cysteine | pH 8.0, 37°C, recombinant mutant Y114F/C116H | Pseudomonas putida | |
1.06 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutant K240N | Pseudomonas putida | |
1.8 | - |
L-methionine | pH 8.0, 37°C, recombinant mutant K240N | Pseudomonas putida | |
2.23 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutant K240I | Pseudomonas putida | |
2.36 | - |
S-methyl-L-cysteine | pH 8.0, 37°C, recombinant wild-type enzyme | Pseudomonas putida | |
4.45 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutant Y114F/C116H | Pseudomonas putida | |
4.83 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutant K240R | Pseudomonas putida | |
5.46 | - |
L-methionine | pH 8.0, 37°C, recombinant mutant K240I | Pseudomonas putida | |
5.53 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutant K240M | Pseudomonas putida | |
6.29 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant K240N | Pseudomonas putida | |
6.8 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant Y114F/C116H | Pseudomonas putida | |
10.4 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant K240I | Pseudomonas putida | |
10.7 | - |
L-cysteine | pH 8.0, 37°C, recombinant wild-type enzyme | Pseudomonas putida | |
20.6 | - |
L-methionine | pH 8.0, 37°C, recombinant mutant K240M | Pseudomonas putida | |
20.6 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant K240M | Pseudomonas putida | |
22 | - |
S-methyl-L-cysteine | pH 8.0, 37°C, recombinant mutant C166H | Pseudomonas putida | |
34.6 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant C166H | Pseudomonas putida | |
37.8 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant mutant K240R | Pseudomonas putida | |
39 | - |
L-methionine | pH 8.0, 37°C, recombinant mutant K240R | Pseudomonas putida | |
64.5 | - |
DL-homocysteine | pH 8.0, 37°C, recombinant wild-type enzyme | Pseudomonas putida | |
66.8 | - |
L-methionine | pH 8.0, 37°C, recombinant wild-type enzyme | Pseudomonas putida | |
77.7 | - |
L-cysteine | pH 8.0, 37°C, recombinant mutant C166H | Pseudomonas putida |