Structural characterization and function determination of a nonspecific carboxylate esterase from the amidohydrolase superfamily with a promiscuous ability to hydrolyze methylphosphonate esters.
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.
Department of Chemistry, Texas A&M University , P.O. Box 30012, College Station, Texas 77842-3012, United States.
Dao Feng
DF
Xiang
Desigan
D
Kumaran
Subramanyam
S
Swaminathan
Frank M
FM
Raushel
D001426:Bacterial Proteins / Q000737:chemistry*; D043182:Carboxylesterase / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004952:Esters; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009943:Organophosphorus Compounds / Q000737:chemistry*; D011487:Protein Conformation; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012242:Rhodobacter sphaeroides / Q000201:enzymology; D013237:Stereoisomerism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004952:Esters; D009943:Organophosphorus Compounds; D021122:Protein Subunits; D011994:Recombinant Proteins; C032627:methylphosphonic acid; D043182:Carboxylesterase