Structural characterization and function determination of a nonspecific carboxylate esterase from the amidohydrolase superfamily with a promiscuous ability to hydrolyze methylphosphonate esters. Academic Article uri icon


  • 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 . The protein folds as a distorted (/)8-barrel, and the subunits associate as a homotetramer. The active site is localized to the C-terminal end of the -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.

published proceedings

  • Biochemistry

author list (cited authors)

  • Xiang, D. F., Kumaran, D., Swaminathan, S., & Raushel, F. M.

citation count

  • 5

complete list of authors

  • Xiang, Dao Feng||Kumaran, Desigan||Swaminathan, Subramanyam||Raushel, Frank M

publication date

  • June 2014