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PMID:24832101
| Citation |
Xiang, DF, Kumaran, D, Swaminathan, S and Raushel, FM (2014) Structural characterization and function determination of a nonspecific carboxylate esterase from the amidohydrolase superfamily with a promiscuous ability to hydrolyze methylphosphonate esters. Biochemistry 53:3476-85 |
|---|---|
| Abstract |
The uncharacterized protein Rsp3690 from Rhodobacter sphaeroides is a member of the amidohydrolase superfamily of enzymes. In this investigation the gene for Rsp3690 was expressed in Escherichia coli and purified to homogeneity, and the three-dimensional structure was determined to a resolution of 1.8 Å. The protein folds as a distorted (β/α)8-barrel, and the subunits associate as a homotetramer. The active site is localized to the C-terminal end of the β-barrel and is highlighted by the formation of a binuclear metal center with two manganese ions that are bridged by Glu-175 and hydroxide. The remaining ligands to the metal center include His-32, His-34, His-207, His-236, and Asp-302. Rsp3690 was shown to catalyze the hydrolysis of a wide variety of carboxylate esters, in addition to organophosphate and organophosphonate esters. The best carboxylate ester substrates identified for Rsp3690 included 2-naphthyl acetate (kcat/Km = 1.0 × 10(5) M(-1) s(-1)), 2-naphthyl propionate (kcat/Km = 1.5 × 10(5) M(-1) s(-1)), 1-naphthyl acetate (kcat/Km = 7.5 × 10(3) M(-1) s(-1)), 4-methylumbelliferyl acetate (kcat/Km = 2.7 × 10(3) M(-1) s(-1)), 4-nitrophenyl acetate (kcat/Km = 2.3 × 10(5) M(-1) s(-1)), and 4-nitrophenyl butyrate (kcat/Km = 8.8 × 10(5) M(-1) s(-1)). The best organophosphonate ester substrates included ethyl 4-nitrophenyl methylphosphonate (kcat/Km = 3.8 × 10(5) M(-1) s(-1)) and isobutyl 4-nitrophenyl methylphosphonate (kcat/Km = 1.1 × 10(4) M(-1) s(-1)). The (SP)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate was hydrolyzed 10 times faster than the less toxic (RP)-enantiomer. The high inherent catalytic activity of Rsp3690 for the hydrolysis of the toxic enantiomer of methylphosphonate esters make this enzyme an attractive target for directed evolution investigations. |
| Links |
PubMed PMC4045322 Online version:10.1021/bi5004266 |
| Keywords |
Bacterial Proteins/chemistry; Carboxylesterase/chemistry; Crystallography, X-Ray; Esters; Hydrolysis; Kinetics; Models, Molecular; Organophosphorus Compounds/chemistry; Protein Conformation; Protein Multimerization; Protein Subunits/chemistry; Recombinant Proteins/chemistry; Rhodobacter sphaeroides/enzymology; Stereoisomerism; Substrate Specificity |
| edit table |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0008081: phosphoric diester hydrolase activity |
ECO:0000314: |
F |
Table 2 shows that Rsp3690 hydrolyzes ethyl 4-nitrophenyl methylphosphonate. |
complete | ||||
| GO:0052689: carboxylic ester hydrolase activity |
ECO:0000314: |
F |
Table two shows that Rsp3690 catalyzes the hydrolysis of the carboxylic ester group of both 2-naphthyl propionate (16) and 4-nitrophenyl butyrate (17). |
complete | ||||
|
enables |
GO:0008081: phosphoric diester hydrolase activity |
ECO:0000314: direct assay evidence used in manual assertion |
F |
Seeded From UniProt |
complete | |||
Notes
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