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Literature summary for 3.1.1.1 extracted from

  • Yan, X.; Wang, J.; Sun, Y.; Zhu, J.; Wu, S.
    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 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.1.1.1

Cloned(Commentary)

Cloned (Comment) Organism
gene mhg, recombinant expression of wild-type and mutant enzymes in Escherichia coli Microbacterium hydrocarbonoxydans

Protein Variants

Protein Variants Comment Organism
L233A site-directed mutagenesis, L233A mutant exhibits esterase activity versus 4-nitrophenyl butyrate Microbacterium hydrocarbonoxydans
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
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
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
L233S site-directed mutagenesis, L233A mutant exhibits esterase activity versus 4-nitrophenyl butyrate Microbacterium hydrocarbonoxydans
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

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten kinetics Microbacterium hydrocarbonoxydans

Organism

Organism UniProt Comment Textmining
Microbacterium hydrocarbonoxydans
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-
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Microbacterium hydrocarbonoxydans NBRC 103074
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Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-naphthyl acetate + H2O substrate of mutant L233G Microbacterium hydrocarbonoxydans 2-naphthol + acetate
-
 ?
2-naphthyl acetate + H2O substrate of mutant L233G Microbacterium hydrocarbonoxydans NBRC 103074 2-naphthol + acetate
-
 ?
4-nitrophenyl butyrate + H2O no activity with wild-type enzyme Mhg but with enzyme L233 mutants Microbacterium hydrocarbonoxydans 4-nitrophenol + butyrate
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 ?
4-nitrophenyl butyrate + H2O no activity with wild-type enzyme Mhg but with enzyme L233 mutants Microbacterium hydrocarbonoxydans NBRC 103074 4-nitrophenol + butyrate
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 ?
4-nitrophenyl caproate + H2O no activity with wild-type enzyme Mhg but with enzyme mutant L233G Microbacterium hydrocarbonoxydans 4-nitrophenol + caproate
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 ?
4-nitrophenyl caproate + H2O no activity with wild-type enzyme Mhg but with enzyme mutant L233G Microbacterium hydrocarbonoxydans NBRC 103074 4-nitrophenol + caproate
-
 ?
5-methyl-2-(propan-2-yl)cyclohexyl acetate + H2O substrate of mutant L233P Microbacterium hydrocarbonoxydans ?
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 ?
5-methyl-2-(propan-2-yl)cyclohexyl acetate + H2O substrate of mutant L233P Microbacterium hydrocarbonoxydans NBRC 103074 ?
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 ?
methyl 2-methylbutyrate + H2O substrate of mutant L233P Microbacterium hydrocarbonoxydans 2-methylbutanoic acid + methanol
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 ?
methyl 2-tetrahydrofuroate + H2O substrate of mutant L233P Microbacterium hydrocarbonoxydans tetrahydrofuran-2-carboxylic acid + methanol
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 ?
methyl 3-cyclohexene-1-carboxylic acid + H2O substrate of mutant L233P Microbacterium hydrocarbonoxydans 3-cyclohexene-1-carboxylic acid + methanol
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 ?
methyl 3-phenyloxirane-2-carboxylate + H2O substrate of mutant L233S Microbacterium hydrocarbonoxydans 3-phenyloxirane-2-carboxylic acid + methanol
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 ?
methyl-(-)-3-(4-methoxyphenyl) oxirane carboxylate + H2O substrate of mutant L233S Microbacterium hydrocarbonoxydans ?
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 ?
additional information enantioselectivity of racemic esters catalyzed by the Mhg mutant variants, mutant substrate specificities, overview Microbacterium hydrocarbonoxydans ?
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 ?
additional information enantioselectivity of racemic esters catalyzed by the Mhg mutant variants, mutant substrate specificities, overview Microbacterium hydrocarbonoxydans NBRC 103074 ?
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 ?

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Microbacterium hydrocarbonoxydans

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Microbacterium hydrocarbonoxydans

General Information

General Information Comment Organism
additional information Mhg homology structure modeling, structure comparison to esterase from Pseudomonas fluorescens, PDB ID 1VA4, overview Microbacterium hydrocarbonoxydans
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