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Literature summary extracted from

  • Adediran, S.A.; Wang, P.F.; Shilabin, A.G.; Baron, C.A.; McLeish, M.J.; Pratt, R.F.
    Specificity and mechanism of mandelamide hydrolase catalysis (2017), Arch. Biochem. Biophys., 618, 23-31 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.1.86 recombinant expression of C-terminally His6-tagged wild-type and mutant enzymes Pseudomonas putida

Protein Variants

EC Number Protein Variants Comment Organism
3.5.1.86 F150L site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type Pseudomonas putida
3.5.1.86 F433A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type Pseudomonas putida
3.5.1.86 F433L site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type Pseudomonas putida
3.5.1.86 K100A site-directed mutagenesis, mutation of a catalytic residue, inactive mutant Pseudomonas putida
3.5.1.86 S180A site-directed mutagenesis, mutation of a catalytic residue, 4200fold decrease in the kcat/Km value Pseudomonas putida
3.5.1.86 S204A site-directed mutagenesis, mutation of a catalytic residue, inactive mutant Pseudomonas putida

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.5.1.86 benzyl (carbamoyloxy)carbamate weak inhibition Pseudomonas putida
3.5.1.86 additional information O-acyl hydroxamates are no irreversible inactivators of MAH but some are found to be transient inhibitors. Reactivity of MAH with some recently studied beta-lactamase inhibitors, overview Pseudomonas putida
3.5.1.86 N-(carbamoyloxy)benzamide
-
Pseudomonas putida
3.5.1.86 O-methoxycarbonylbenzohydroxamate
-
Pseudomonas putida
3.5.1.86 phenyl carbamate
-
Pseudomonas putida
3.5.1.86 phenylmethylboronic acid a potent inhibitor of enzyme MAH that forms a transition state analogue structure with the enzyme Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.1.86 0.009
-
(S)-mandelamide recombinant mutant F150W, pH 7.8, 25°C Pseudomonas putida
3.5.1.86 0.018
-
(S)-mandelamide recombinant wild-type enzyme, pH 7.8, 25°C Pseudomonas putida
3.5.1.86 0.043
-
(S)-mandelamide recombinant mutant F433W, pH 7.8, 25°C Pseudomonas putida
3.5.1.86 0.135
-
(S)-mandelamide recombinant mutant F433L, pH 7.8, 25°C Pseudomonas putida
3.5.1.86 1.53
-
(S)-mandelamide recombinant mutant F433A, pH 7.8, 25°C Pseudomonas putida

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.5.1.86 (R)-mandelamide + H2O Pseudomonas putida
-
(R)-mandelate + NH3
-
?
3.5.1.86 (S)-mandelamide + H2O Pseudomonas putida
-
(S)-mandelate + NH3
-
?

Organism

EC Number Organism UniProt Comment Textmining
3.5.1.86 Pseudomonas putida Q84DC4 i.e. Arthrobacter siderocapsulatus
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.1.86 (R)-mandelamide + H2O
-
Pseudomonas putida (R)-mandelate + NH3
-
?
3.5.1.86 (S)-mandelamide + H2O
-
Pseudomonas putida (S)-mandelate + NH3
-
?
3.5.1.86 4-nitroacetanilide + H2O
-
Pseudomonas putida ?
-
?
3.5.1.86 4-nitrophenylacetamide + H2O
-
Pseudomonas putida 4-nitrophenol + acetamide
-
?
3.5.1.86 4-nitrophenylacetate + H2O
-
Pseudomonas putida 4-nitrophenol + acetate
-
?
3.5.1.86 4-nitrophenylcarbonate + H2O
-
Pseudomonas putida 4-nitrophenol + CO2
-
?
3.5.1.86 methyl 4-nitrophenylcarbonate + H2O
-
Pseudomonas putida ?
-
?
3.5.1.86 methyl mandelate + H2O
-
Pseudomonas putida ?
-
?
3.5.1.86 additional information the lack of reactivity of MAH with the corresponding ethyl ester confirms the very limited size of the MAH leaving group site. The reactivity of MAH with 4-nitrophenyl acetate and methyl 4-nitrophenyl carbonate, therefore, suggests formation of an inverse acyl-enzyme where the small acyl-group occupies the normal leaving group site. Both (R)- and (S)-mandelamide are excellent substrates for MAH, while the enzyme can hydrolyze phenylacetamide with even higher catalytic efficiency. Reaction scheme for interaction of substrates with MAH. Substrate specificity, overview. No activity with ethyl 4-nitrophenylacetate, phenyl carbamate, and N-benzyloxycarbonyl-glycinamide, poor activity with phenyl acetate, 4-nitrophenyl n-butyrate, and ethyl 4-nitrophenyl carbonate Pseudomonas putida ?
-
-
3.5.1.86 N-benzyloxycarbonylglycinamide + H2O
-
Pseudomonas putida ?
-
?
3.5.1.86 phenyl carbamate + H2O
-
Pseudomonas putida ?
-
?
3.5.1.86 phenylacetamide + H2O
-
Pseudomonas putida phenylacetate + NH3
-
?

Synonyms

EC Number Synonyms Comment Organism
3.5.1.86 MAH
-
Pseudomonas putida
3.5.1.86 mandelamide hydrolase
-
Pseudomonas putida

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.1.86 25
-
assay at Pseudomonas putida

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.5.1.86 20
-
(S)-mandelamide recombinant mutant F433A, pH 7.8, 25°C Pseudomonas putida
3.5.1.86 43
-
(S)-mandelamide recombinant mutant F150W, pH 7.8, 25°C Pseudomonas putida
3.5.1.86 90
-
(S)-mandelamide recombinant mutant F433W, pH 7.8, 25°C Pseudomonas putida
3.5.1.86 105
-
(S)-mandelamide recombinant mutant F433L, pH 7.8, 25°C Pseudomonas putida
3.5.1.86 150
-
(S)-mandelamide recombinant wild-type enzyme, pH 7.8, 25°C Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.5.1.86 7.8
-
assay at Pseudomonas putida

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
3.5.1.86 0.000025
-
O-methoxycarbonylbenzohydroxamate pH 7.8, 25°C Pseudomonas putida
3.5.1.86 0.000025
-
phenyl carbamate pH 7.8, 25°C Pseudomonas putida
3.5.1.86 0.000027
-
phenylmethylboronic acid pH 7.8, 25°C Pseudomonas putida
3.5.1.86 1.4
-
benzyl (carbamoyloxy)carbamate pH 7.8, 25°C Pseudomonas putida

General Information

EC Number General Information Comment Organism
3.5.1.86 additional information the catalytic triad of MAH is composed of Ser204, Ser180, and Lys100. Modeling of substrate binding. A model of the deacylation tetrahedral intermediate is derived from reaction of MAH with phenylacetamide, 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
3.5.1.86 13.1
-
(S)-mandelamide recombinant mutant F433A, pH 7.8, 25°C Pseudomonas putida
3.5.1.86 777.8
-
(S)-mandelamide recombinant mutant F433L, pH 7.8, 25°C Pseudomonas putida
3.5.1.86 2093
-
(S)-mandelamide recombinant mutant F433W, pH 7.8, 25°C Pseudomonas putida
3.5.1.86 4778
-
(S)-mandelamide recombinant mutant F150W, pH 7.8, 25°C Pseudomonas putida
3.5.1.86 8333
-
(S)-mandelamide recombinant wild-type enzyme, pH 7.8, 25°C Pseudomonas putida