(R)-and (S)-2-hydroxypropyl-CoM (R-HPC and S-HPC) are produced as intermediates in bacterial propylene metabolism from the nucleophilic addition of coenzyme M to (R)-and (S)-epoxypropane, respectively. Two highly enantioselective dehydrogenases (R-HPCDH and S-HPCDH) belonging to the short-chain dehydrogenase/reductase family catalyze the conversion of R-HPC and S-HPC to 2-ketopropyl-CoM (2-KPC), which undergoes reductive cleavage and carboxylation to produce acetoacetate. In the present study, one of three copies of S-HPCDH enzymes present on a linear megaplasmid in Xanthobacter autotrophicus strain Py2 has been cloned and overexpressed, allowing the first detailed side by side characterization of the R-HPCDH and S-HPCDH enzymes. The catalytic triad of S-HPCDH was found to consist of Y156, K160, and S143. R211 and K214 were identified as the amino acid residues coordinating the sulfonate of CoM in S-HPC. R211A and K214A mutants were severely impaired in the oxidation of R-HPC or reduction of 2-KPC but were largely unaffected in the oxidation and reduction of aliphatic alcohols and ketones. Kinetic analyses using (R)-and (S)-HPC as substrates revealed that enantioselectivity in R-HPCDH (value, 944) was dictated largely by differences in k cat while enantioselectivity for S-HPCDH (value, 658) was dictated largely by changes in K m . S-HPCDH had an inherent high enantioselectivity for producing (S)-2-butanol from 2-butanone that was unaffected by modulators that interact with the sulfonate binding site. The tertiary alcohol 2-methyl-2-hydroxypropyl-CoM (M-HPC) was a competitive inhibitor of R-HPCDH-catalyzed R-HPC oxidation, with a K is similar to the K m for R-HPC, but was not an inhibitor of S-HPCDH. The primary alcohol 2-hydroxyethyl-CoM was a substrate for both R-HPCDH and S-HPCDH with identical K m values. The pH dependence of kinetic parameters suggests that the hydroxyl group is † This work was supported by National Institutes of Health Grant GM51805 to S.AE. and by Department of Energy Grant DE-FG02-04ER15563 to J.W.P. * To whom correspondence should be addressed: (435) 797-3969 (phone); (435) 797-3390 (fax); scott.ensign@usu.edu.
Abbreviations: R-HPC, 2-[(R)-2-hydroxypropylthio]ethanesulfonate, (R)-2-hydroxypropyl-CoM); S-HPC, 2-[(S)-2-hydroxypropylthio] ethanesulfonate, (S)-2-hydroxypropyl-CoM); S-HPCDH, 2-[(S)-2-hydroxypropylthio]ethanesulfonate dehydrogenase; rS-HPCDH, recombinant 2-[(S)-2-hydroxypropylthio]ethanesulfonate dehydrogenase; R-HPCDH, 2-[(R)-2-hydroxypropylthio]ethanesulfonate dehydrogenase; rR-HPCDH, recombinant 2-[(R)-2-hydroxypropylthio]ethanesulfonatedehydrogenase; M-HPC, 2-(2-methyl-2-hydroxypropylthio)ethanesulfonate; HEC, 2-(2-hydroxyethylthio)ethanesulfonate; 2-KPC, 2-(2-ketopropylthio)ethanesulfonate (2-ketopropyl-CoM); 2-KPCC, 2-(2-ketopropylthio)ethanesulfonate carboxylase/oxidoreductase; CoM, coenzyme M, (2-mercaptoethanesulfonate); SDR, short-chain dehydrogenase/reductase; Tris, tris(hydroxymethyl)aminomethane, CD, circular dichroism; ee, enantiomeric excess; E, enantio...