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Literature summary extracted from

  • Fukumoto, M.; Kudou, D.; Murano, S.; Shiba, T.; Sato, D.; Tamura, T.; Harada, S.; Inagaki, K.
    The role of amino acid residues in the active site of L-methionine gamma-lyase from Pseudomonas putida (2012), Biosci. Biotechnol. Biochem., 76, 1275-1284.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.4.1.11 recombinant expression of wild-type and mutant enzymes in Escherichia coli strain HB101 Pseudomonas putida

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.4.1.11 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

EC Number Protein Variants Comment Organism
4.4.1.11 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
4.4.1.11 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
4.4.1.11 D241E site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme Pseudomonas putida
4.4.1.11 D241H site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme Pseudomonas putida
4.4.1.11 D241I site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme Pseudomonas putida
4.4.1.11 D241K site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme Pseudomonas putida
4.4.1.11 D241M site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme Pseudomonas putida
4.4.1.11 D241N site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme Pseudomonas putida
4.4.1.11 D241R site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme Pseudomonas putida
4.4.1.11 D241T site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme Pseudomonas putida
4.4.1.11 K240D site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme Pseudomonas putida
4.4.1.11 K240E site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme Pseudomonas putida
4.4.1.11 K240H site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme Pseudomonas putida
4.4.1.11 K240M site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme Pseudomonas putida
4.4.1.11 K240R site-directed mutagenesis, the mutant shows reduced activity in the elimination reaction compared to the wild-type enzyme Pseudomonas putida
4.4.1.11 K240S site-directed mutagenesis Pseudomonas putida

Inhibitors

EC Number Inhibitors Comment Organism Structure
4.4.1.11 DL-propargylglycine inhibition profiles of wild-type and mutant enzymes, overview Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.4.1.11 0.04
-
 L-methionine pH 8.0, 37°C, recombinant mutant K240R Pseudomonas putida
4.4.1.11 0.2
-
 L-cysteine pH 8.0, 37°C, recombinant wild-type enzyme Pseudomonas putida
4.4.1.11 0.2
-
 L-cysteine pH 8.0, 37°C, recombinant mutant Y114F/C116H Pseudomonas putida
4.4.1.11 0.2
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant Y114F/C116H Pseudomonas putida
4.4.1.11 0.3
-
 L-cysteine pH 8.0, 37°C, recombinant mutant C166H Pseudomonas putida
4.4.1.11 0.3
-
 L-cysteine pH 8.0, 37°C, recombinant mutants K240R and K240M Pseudomonas putida
4.4.1.11 0.4
-
 S-methyl-L-cysteine pH 8.0, 37°C, recombinant mutant Y114F/C116H Pseudomonas putida
4.4.1.11 0.5
-
 L-methionine pH 8.0, 37°C, recombinant wild-type enzyme Pseudomonas putida
4.4.1.11 0.6
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant K240R Pseudomonas putida
4.4.1.11 0.7
-
 S-methyl-L-cysteine pH 8.0, 37°C, recombinant wild-type enzyme Pseudomonas putida
4.4.1.11 0.8
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant K240N Pseudomonas putida
4.4.1.11 1
-
 L-cysteine pH 8.0, 37°C, recombinant mutants K240I and K240N Pseudomonas putida
4.4.1.11 1.1
-
 DL-homocysteine pH 8.0, 37°C, recombinant wild-type enzyme Pseudomonas putida
4.4.1.11 1.4
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant C166H Pseudomonas putida
4.4.1.11 1.4
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant K240M Pseudomonas putida
4.4.1.11 2.1
-
 S-methyl-L-cysteine pH 8.0, 37°C, recombinant mutant C166H Pseudomonas putida
4.4.1.11 2.3
-
 L-methionine pH 8.0, 37°C, recombinant mutant C166H Pseudomonas putida
4.4.1.11 4.8
-
 L-methionine pH 8.0, 37°C, recombinant mutant K240I Pseudomonas putida
4.4.1.11 5.7
-
 L-methionine pH 8.0, 37°C, recombinant mutant K240M Pseudomonas putida
4.4.1.11 6
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant K240I Pseudomonas putida
4.4.1.11 9.8
-
 L-methionine pH 8.0, 37°C, recombinant mutant K240N Pseudomonas putida

Organism

EC Number Organism UniProt Comment Textmining
4.4.1.11 Pseudomonas putida P13254
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.4.1.11 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 and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.4.1.11 DL-homocysteine + 2-mercaptoethanol gamma-replacement reaction, wild-type enzyme Pseudomonas putida H2S + S-(beta-hydroxyethyl)-L-homocysteine
-
 ?
4.4.1.11 L-cysteine + 2-mercaptoethanol beta-replacement reaction, wild-type enzyme and mutant C116H Pseudomonas putida H2S + S-(beta-hydroxyethyl)-L-cysteine
-
 ?
4.4.1.11 L-methionine + 2-mercaptoethanol gamma-replacement reaction, wild-type enzyme Pseudomonas putida methanethiol + S-(beta-hydroxyethyl)-L-homocysteine
-
 ?
4.4.1.11 S-methyl-L-cysteine + 2-mercaptoethanol
-
 Pseudomonas putida ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
4.4.1.11 L-methionine gamma-lyase
-
 Pseudomonas putida
4.4.1.11 MGL
-
 Pseudomonas putida

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.4.1.11 37
-
 assay at Pseudomonas putida

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
4.4.1.11 50
-
 purified wild-type enzyme and mutants, all completely stable, except for mutants D241H and D241I loosing about 10% activity Pseudomonas putida
4.4.1.11 70
-
 purified mutant D241R, loss of 70% activity Pseudomonas putida
4.4.1.11 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
4.4.1.11 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 [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.4.1.11 0.03
-
 L-methionine pH 8.0, 37°C, recombinant mutant C166H Pseudomonas putida
4.4.1.11 0.12
-
 S-methyl-L-cysteine pH 8.0, 37°C, recombinant mutant Y114F/C116H Pseudomonas putida
4.4.1.11 0.89
-
 L-cysteine pH 8.0, 37°C, recombinant mutant Y114F/C116H Pseudomonas putida
4.4.1.11 1.06
-
 L-cysteine pH 8.0, 37°C, recombinant mutant K240N Pseudomonas putida
4.4.1.11 1.36
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant Y114F/C116H Pseudomonas putida
4.4.1.11 1.45
-
 L-cysteine pH 8.0, 37°C, recombinant mutant K240R Pseudomonas putida
4.4.1.11 1.58
-
 S-methyl-L-cysteine pH 8.0, 37°C, recombinant wild-type enzyme Pseudomonas putida
4.4.1.11 1.66
-
 L-cysteine pH 8.0, 37°C, recombinant mutant K240M Pseudomonas putida
4.4.1.11 2.13
-
 L-cysteine pH 8.0, 37°C, recombinant wild-type enzyme Pseudomonas putida
4.4.1.11 2.23
-
 L-cysteine pH 8.0, 37°C, recombinant mutant K240I Pseudomonas putida
4.4.1.11 5.03
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant K240N Pseudomonas putida
4.4.1.11 22.7
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant K240R Pseudomonas putida
4.4.1.11 23.3
-
 L-cysteine pH 8.0, 37°C, recombinant mutant C166H Pseudomonas putida
4.4.1.11 28.8
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant K240M Pseudomonas putida
4.4.1.11 33.4
-
 L-methionine pH 8.0, 37°C, recombinant wild-type enzyme Pseudomonas putida
4.4.1.11 46.3
-
 S-methyl-L-cysteine pH 8.0, 37°C, recombinant mutant C166H Pseudomonas putida
4.4.1.11 48.4
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant C166H Pseudomonas putida
4.4.1.11 62.2
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant K240I Pseudomonas putida
4.4.1.11 71
-
 DL-homocysteine pH 8.0, 37°C, recombinant wild-type enzyme Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.4.1.11 8
-
 assay at Pseudomonas putida

Cofactor

EC Number Cofactor Comment Organism Structure
4.4.1.11 pyridoxal 5'-phosphate
-
 Pseudomonas putida

General Information

EC Number General Information Comment Organism
4.4.1.11 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 [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
4.4.1.11 0.01
-
 L-methionine pH 8.0, 37°C, recombinant mutant C166H Pseudomonas putida
4.4.1.11 0.3
-
 S-methyl-L-cysteine pH 8.0, 37°C, recombinant mutant Y114F/C116H Pseudomonas putida
4.4.1.11 1.06
-
 L-cysteine pH 8.0, 37°C, recombinant mutant K240N Pseudomonas putida
4.4.1.11 1.8
-
 L-methionine pH 8.0, 37°C, recombinant mutant K240N Pseudomonas putida
4.4.1.11 2.23
-
 L-cysteine pH 8.0, 37°C, recombinant mutant K240I Pseudomonas putida
4.4.1.11 2.36
-
 S-methyl-L-cysteine pH 8.0, 37°C, recombinant wild-type enzyme Pseudomonas putida
4.4.1.11 4.45
-
 L-cysteine pH 8.0, 37°C, recombinant mutant Y114F/C116H Pseudomonas putida
4.4.1.11 4.83
-
 L-cysteine pH 8.0, 37°C, recombinant mutant K240R Pseudomonas putida
4.4.1.11 5.46
-
 L-methionine pH 8.0, 37°C, recombinant mutant K240I Pseudomonas putida
4.4.1.11 5.53
-
 L-cysteine pH 8.0, 37°C, recombinant mutant K240M Pseudomonas putida
4.4.1.11 6.29
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant K240N Pseudomonas putida
4.4.1.11 6.8
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant Y114F/C116H Pseudomonas putida
4.4.1.11 10.4
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant K240I Pseudomonas putida
4.4.1.11 10.7
-
 L-cysteine pH 8.0, 37°C, recombinant wild-type enzyme Pseudomonas putida
4.4.1.11 20.6
-
 L-methionine pH 8.0, 37°C, recombinant mutant K240M Pseudomonas putida
4.4.1.11 20.6
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant K240M Pseudomonas putida
4.4.1.11 22
-
 S-methyl-L-cysteine pH 8.0, 37°C, recombinant mutant C166H Pseudomonas putida
4.4.1.11 34.6
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant C166H Pseudomonas putida
4.4.1.11 37.8
-
 DL-homocysteine pH 8.0, 37°C, recombinant mutant K240R Pseudomonas putida
4.4.1.11 39
-
 L-methionine pH 8.0, 37°C, recombinant mutant K240R Pseudomonas putida
4.4.1.11 64.5
-
 DL-homocysteine pH 8.0, 37°C, recombinant wild-type enzyme Pseudomonas putida
4.4.1.11 66.8
-
 L-methionine pH 8.0, 37°C, recombinant wild-type enzyme Pseudomonas putida
4.4.1.11 77.7
-
 L-cysteine pH 8.0, 37°C, recombinant mutant C166H Pseudomonas putida