Molecular Basis for Enantioselectivity in the (R)- and (S)-Hydroxypropylthioethanesulfonate Dehydrogenases, a Unique Pair of Stereoselective Short-Chain Dehydrogenases/Reductases Involved in Aliphatic Epoxide Carboxylation

摘要

(R)- and (S)-2-hydroxypropyl-CoM (R-HPC and S-HPC) are produced as intermediates innbacterial propylenemetabolismfromthe nucleophilic addition of coenzymeMto (R)- and (S)-epoxypropane,nrespectively. Two highly enantioselective dehydrogenases (R-HPCDH and S-HPCDH) belonging to thenshort-chain dehydrogenase/reductase family catalyze the conversion of R-HPC and S-HPC to 2-ketopropyl-nCoM(2-KPC), which undergoes reductive cleavage and carboxylation to produce acetoacetate. In the presentnstudy, one of three copies of S-HPCDH enzymes present on a linear megaplasmid in Xanthobacternautotrophicus strain Py2 has been cloned and overexpressed, allowing the first detailed side by sidencharacterization of the R-HPCDH and S-HPCDH enzymes. The catalytic triad of S-HPCDH was foundnto consist of Y156, K160, and S143. R211 and K214 were identified as the amino acid residues coordinatingnthe sulfonate of CoM in S-HPC. R211A and K214A mutants were severely impaired in the oxidation of S-nHPC or reduction of 2-KPC but were largely unaffected in the oxidation and reduction of aliphatic alcoholsnand ketones.Kinetic analyses using R- and S-HPC as substrates revealed that enantioselectivity in R-HPCDHn(value, 944) was dictated largely by differences in kcat while enantioselectivity for S-HPCDH(value, 1315) wasndictated largely by changes in Km. S-HPCDH had an inherent high enantioselectivity for producing (S)-2-nbutanol from2-butanone that was unaffected bymodulators that interact with the sulfonate binding site. Thentertiary alcohol 2-methyl-2-hydroxypropyl-CoM (M-HPC) was a competitive inhibitor of R-HPCDH-ncatalyzed R-HPC oxidation, with a Kis similar to the Km for R-HPC, but was not an inhibitor of S-HPCDH.nThe primary alcohol 2-hydroxyethyl-CoMwas a substrate for both R-HPCDHand S-HPCDHwith identicalnKm values. The pH dependence of kinetic parameters suggests that the hydroxyl group is a larger contributornto S-HPC binding to S-HPCDH than for R-HPC binding to R-HPCDH. It is proposed that active sitenconstraints within the S-HPCDHprevent proper binding of R-HPC andM-HPC due to steric clashes with thenimproperly aligned methyl group on the C2 carbon, resulting in a different mechanism for controllingnsubstrate specificity and enantioselectivity than present in the R-HPCDH.