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.