Any feedback?
Literature summary extracted from
- Facilitating the evolution of esterase activity from a promiscuous enzyme (Mhg) with catalytic functions of amide hydrolysis and carboxylic acid perhydrolysis by engineering the substrate entrance tunnel (2016), Appl. Environ. Microbiol., 82, 6748-6756 .
Application
| EC Number | Application | Comment | Organism |
|---|---|---|---|
| 3.5.2.B3 | synthesis | Mhg can be utilized to prepare chiral gamma-lactam, which is an important chiral intermediate for the synthesis of a series of antiviral drugs, such as abacavir (targeting HIV) and peramivir (targeting hepatitis and pandemic influenza viruses) | Microbacterium hydrocarbonoxydans |
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 3.1.1.1 | gene mhg, recombinant expression of wild-type and mutant enzymes in Escherichia coli | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | gene mhg, recombinant expression of C-terminally His-tagged wild-type and mutant enzymes in Escherichia coli TOP10 cells | Microbacterium hydrocarbonoxydans |
Protein Variants
| EC Number | Protein Variants | Comment | Organism |
|---|---|---|---|
| 3.1.1.1 | L233A | site-directed mutagenesis, L233A mutant exhibits esterase activity versus 4-nitrophenyl butyrate | Microbacterium hydrocarbonoxydans |
| 3.1.1.1 | L233A/F144R | site-directed mutagenesis, the mutant is a very specific esterase without any beta-lactamase and perhydrolase activities, and almost no (-)-gamma-lactamase activity | Microbacterium hydrocarbonoxydans |
| 3.1.1.1 | L233G | site-directed mutagenesis, the mutant is a very specific esterase without any beta-lactamase and perhydrolase activities, and almost no (-)-gamma-lactamase activity | Microbacterium hydrocarbonoxydans |
| 3.1.1.1 | L233P | site-directed mutagenesis, L233A mutant exhibits esterase activity versus 4-nitrophenyl butyrate and shows slightly reduced (-)-gamma-lactamase activity compared to the wild-type | Microbacterium hydrocarbonoxydans |
| 3.1.1.1 | L233S | site-directed mutagenesis, L233A mutant exhibits esterase activity versus 4-nitrophenyl butyrate | Microbacterium hydrocarbonoxydans |
| 3.1.1.1 | additional information | by engineering the entrance tunnel, entering into the substrate-binding pocket, with only one or two amino acid substitutions, five esterase variants of Mhg are obtained. The variants exhibit a very broad substrate acceptance, hydrolyzing not only the classical 4-nitrophenol esters but also various types of chiral esters, which are widely used as drug intermediates. Site 233 at the entrance tunnel of Mhg is found to play a pivotal role in modulating the three catalytic activities by adjusting the size and shape of the tunnel, with different amino acid substitutions at this site facilitating different activities. Remarkably, the variant with the L233G mutant is a very specific esterase without any beta-lactamase and perhydrolase activities. Considering the amino acid conservation and differentiation, this site could be a key target for future protein engineering. Engineering the entrance tunnel is an efficient strategy to regulate enzyme catalytic capabilities | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | F144A | site-directed mutagenesis | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | F162A | site-directed mutagenesis | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | I232A | site-directed mutagenesis | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | L233A | site-directed mutagenesis, L233A mutant exhibits esterase activity versus 4-nitrophenyl butyrate and shows reduced (-)-gamma-lactamase activity compared to the wild-type | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | L233A/F144R | site-directed mutagenesis, the mutant is a very specific esterase without any beta-lactamase and perhydrolase activities, and almost no (-)-gamma-lactamase activity | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | L233C | site-directed mutagenesis, the mutant shows highly increased perhydrolase activity compared to wild-type, and similar (-)-gamma-lactamase activity, and no esterase activity | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | L233G | site-directed mutagenesis, the mutant is a very specific esterase without any beta-lactamase and perhydrolase activities, and almost no (-)-gamma-lactamase activity | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | L233M | site-directed mutagenesis, the mutant shows highly increased perhydrolase activity compared to wild-type, but no (-)-gamma-lactamase activity and no esterase activity | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | L233P | site-directed mutagenesis, L233P mutant exhibits esterase activity versus 4-nitrophenyl butyrate and shows slightly reduced (-)-gamma-lactamase activity compared to the wild-type | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | L233S | site-directed mutagenesis, L233S mutant exhibits esterase activity versus 4-nitrophenyl butyrate and shows reduced (-)-gamma-lactamase activity compared to the wild-type | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | L233T | site-directed mutagenesis, the mutant shows similar (-)-gamma-lactamase activity compared to wild-type, and no perhydrolase and esterase activities | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | additional information | by engineering the entrance tunnel, entering into the substrate-binding pocket, with only one or two amino acid substitutions, five esterase variants of Mhg are obtained. The variants exhibit a very broad substrate acceptance, hydrolyzing not only the classical 4-nitrophenol esters but also various types of chiral esters, which are widely used as drug intermediates. Site 233 at the entrance tunnel of Mhg is found to play a pivotal role in modulating the three catalytic activities by adjusting the size and shape of the tunnel, with different amino acid substitutions at this site facilitating different activities. Remarkably, the variant with the L233G mutant is a very specific esterase without any beta-lactamase and perhydrolase activities. Considering the amino acid conservation and differentiation, this site could be a key target for future protein engineering. Engineering the entrance tunnel is an efficient strategy to regulate enzyme catalytic capabilities | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | W204A | site-directed mutagenesis | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | Y32A | site-directed mutagenesis | Microbacterium hydrocarbonoxydans |
KM Value [mM]
| EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 3.1.1.1 | additional information | - |
additional information | Michaelis-Menten kinetics | Microbacterium hydrocarbonoxydans | |
| 3.5.2.B3 | additional information | - |
additional information | Michaelis-Menten kinetics | Microbacterium hydrocarbonoxydans |
Natural Substrates/ Products (Substrates)
| EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 3.5.2.B3 | a (-)-gamma-lactam + H2O | Microbacterium hydrocarbonoxydans | wild-type enzyme | a substituted gamma-amino acid | - |
? |  |
| 3.5.2.B3 | a (-)-gamma-lactam + H2O | Microbacterium hydrocarbonoxydans NBRC 103074 | wild-type enzyme | a substituted gamma-amino acid | - |
? | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 3.1.1.1 | Microbacterium hydrocarbonoxydans | - |
- |
- |
| 3.1.1.1 | Microbacterium hydrocarbonoxydans NBRC 103074 | - |
- |
- |
| 3.5.2.B3 | Microbacterium hydrocarbonoxydans | E3SVS0 | - |
- |
| 3.5.2.B3 | Microbacterium hydrocarbonoxydans NBRC 103074 | E3SVS0 | - |
- |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 3.1.1.1 | 2-naphthyl acetate + H2O | substrate of mutant L233G | Microbacterium hydrocarbonoxydans | 2-naphthol + acetate | - |
? | |
| 3.1.1.1 | 2-naphthyl acetate + H2O | substrate of mutant L233G | Microbacterium hydrocarbonoxydans NBRC 103074 | 2-naphthol + acetate | - |
? | |
| 3.1.1.1 | 4-nitrophenyl butyrate + H2O | no activity with wild-type enzyme Mhg but with enzyme L233 mutants | Microbacterium hydrocarbonoxydans | 4-nitrophenol + butyrate | - |
? | |
| 3.1.1.1 | 4-nitrophenyl butyrate + H2O | no activity with wild-type enzyme Mhg but with enzyme L233 mutants | Microbacterium hydrocarbonoxydans NBRC 103074 | 4-nitrophenol + butyrate | - |
? | |
| 3.1.1.1 | 4-nitrophenyl caproate + H2O | no activity with wild-type enzyme Mhg but with enzyme mutant L233G | Microbacterium hydrocarbonoxydans | 4-nitrophenol + caproate | - |
? | |
| 3.1.1.1 | 4-nitrophenyl caproate + H2O | no activity with wild-type enzyme Mhg but with enzyme mutant L233G | Microbacterium hydrocarbonoxydans NBRC 103074 | 4-nitrophenol + caproate | - |
? | |
| 3.1.1.1 | 5-methyl-2-(propan-2-yl)cyclohexyl acetate + H2O | substrate of mutant L233P | Microbacterium hydrocarbonoxydans | ? | - |
? | |
| 3.1.1.1 | 5-methyl-2-(propan-2-yl)cyclohexyl acetate + H2O | substrate of mutant L233P | Microbacterium hydrocarbonoxydans NBRC 103074 | ? | - |
? | |
| 3.1.1.1 | methyl 2-methylbutyrate + H2O | substrate of mutant L233P | Microbacterium hydrocarbonoxydans | 2-methylbutanoic acid + methanol | - |
? | |
| 3.1.1.1 | methyl 2-tetrahydrofuroate + H2O | substrate of mutant L233P | Microbacterium hydrocarbonoxydans | tetrahydrofuran-2-carboxylic acid + methanol | - |
? | |
| 3.1.1.1 | methyl 3-cyclohexene-1-carboxylic acid + H2O | substrate of mutant L233P | Microbacterium hydrocarbonoxydans | 3-cyclohexene-1-carboxylic acid + methanol | - |
? | |
| 3.1.1.1 | methyl 3-phenyloxirane-2-carboxylate + H2O | substrate of mutant L233S | Microbacterium hydrocarbonoxydans | 3-phenyloxirane-2-carboxylic acid + methanol | - |
? | |
| 3.1.1.1 | methyl-(-)-3-(4-methoxyphenyl) oxirane carboxylate + H2O | substrate of mutant L233S | Microbacterium hydrocarbonoxydans | ? | - |
? | |
| 3.1.1.1 | additional information | enantioselectivity of racemic esters catalyzed by the Mhg mutant variants, mutant substrate specificities, overview | Microbacterium hydrocarbonoxydans | ? | - |
? | |
| 3.1.1.1 | additional information | enantioselectivity of racemic esters catalyzed by the Mhg mutant variants, mutant substrate specificities, overview | Microbacterium hydrocarbonoxydans NBRC 103074 | ? | - |
? | |
| 3.5.2.B3 | (-)-2-azabicyclo[2.2.1]hept-5-en-3-one [(-)-gamma-lactam] + H2O | - |
Microbacterium hydrocarbonoxydans | ? | - |
? | |
| 3.5.2.B3 | (-)-2-azabicyclo[2.2.1]hept-5-en-3-one [(-)-gamma-lactam] + H2O | - |
Microbacterium hydrocarbonoxydans NBRC 103074 | ? | - |
? | |
| 3.5.2.B3 | a (-)-gamma-lactam + H2O | wild-type enzyme | Microbacterium hydrocarbonoxydans | a substituted gamma-amino acid | - |
? | |
| 3.5.2.B3 | a (-)-gamma-lactam + H2O | wild-type enzyme | Microbacterium hydrocarbonoxydans NBRC 103074 | a substituted gamma-amino acid | - |
? | |
| 3.5.2.B3 | additional information | the enzyme is also active as esterase with methyl phenyl glycidate, methyl-(-)-3-(4-methoxyphenyl) oxirane carboxylate, methyl 2-tetrahydrofuroate, 3-cyclohexene-1-carboxylic acid, and methyl 2-methylbutyrate, cf. EC 3.1.1.1. No activity of the wild-type enzyme Mhg with 4-nitrophenol butyrate, 4-nitrophenyl caproate, and 1-naphthyl acetate, but high activity with enzyme mutants L233G, L233S, L233P, L233A, and L233A/F144R | Microbacterium hydrocarbonoxydans | ? | - |
? | |
| 3.5.2.B3 | additional information | the enzyme is also active as esterase with methyl phenyl glycidate, methyl-(-)-3-(4-methoxyphenyl) oxirane carboxylate, methyl 2-tetrahydrofuroate, 3-cyclohexene-1-carboxylic acid, and methyl 2-methylbutyrate, cf. EC 3.1.1.1. No activity of the wild-type enzyme Mhg with 4-nitrophenol butyrate, 4-nitrophenyl caproate, and 1-naphthyl acetate, but high activity with enzyme mutants L233G, L233S, L233P, L233A, and L233A/F144R | Microbacterium hydrocarbonoxydans NBRC 103074 | ? | - |
? | |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 3.5.2.B3 | mHG | - |
Microbacterium hydrocarbonoxydans |
Temperature Optimum [°C]
| EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 3.1.1.1 | 30 | - |
assay at | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | 30 | - |
assay at | Microbacterium hydrocarbonoxydans |
pH Optimum
| EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|---|
| 3.1.1.1 | 7.5 | - |
assay at | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | 7.5 | - |
assay at | Microbacterium hydrocarbonoxydans |
General Information
| EC Number | General Information | Comment | Organism |
|---|---|---|---|
| 3.1.1.1 | additional information | Mhg homology structure modeling, structure comparison to esterase from Pseudomonas fluorescens, PDB ID 1VA4, overview | Microbacterium hydrocarbonoxydans |
| 3.1.1.1 | physiological function | despite having high structural similarity to and sharing an identical catalytic triad with an extensively studied esterase from Pseudomonas fluorescens, wild-type enzyme Mhg from Microbacterium hydrocarbonoxydans does not show any esterase activity. Mhg, a typical alpha/beta fold hydrolase, is a gamma-lactamase (EC 3.5.2.) and also shows perhydrolase activities. But the engineered residue L233 point mutants show specific esterase activities, overview | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | additional information | Mhg homology structure modeling, structure comparison to esterase from Pseudomonas fluorescens, PDB ID 1VA4, molecular docking, overview | Microbacterium hydrocarbonoxydans |
| 3.5.2.B3 | physiological function | despite having high structural similarity to and sharing an identical catalytic triad with an extensively studied esterase from Pseudomonas fluorescens, wild-type enzyme Mhg from Microbacterium hydrocarbonoxydans does not show any esterase activity. Mhg, a typical alpha/beta fold hydrolase, is a gamma-lactamase (EC 3.5.2.) and also shows perhydrolase activities. But the engineered residue L233 point mutants show specific esterase activities, overview | Microbacterium hydrocarbonoxydans |
Use of this online version of BRENDA is free under the CC BY 4.0 license. See terms of use for full details.
Release 2023.1 (January 2023)
Legal
Curated at
Member of
