RESUMEN
(R)- and (S)-hydroxypropyl-coenzyme M dehydrogenases (R- and S-HPCDH) are stereospecific enzymes that are central to the metabolism of propylene and epoxide in Xanthobacter autotrophicus. The bacterium produces R- and S-HPCDH simultaneously to facilitate transformation of R- and S-enantiomers of epoxypropane to a common achiral product 2-ketopropyl-CoM (2-KPC). Both R- and S-HPCDH are highly specific for their respective substrates as each enzyme displays less than 0.5% activity with the opposite substrate isomer. In order to elucidate the structural basis for stereospecificity displayed by R- and S-HPCDH we have determined substrate bound crystal structures of S-HPCDH to 1.6Å resolution. Comparisons to the previously reported product-bound structure of R-HPCDH reveal that although the placement of catalytic residues within the active site of each enzyme is nearly identical, structural differences in the surrounding area provide each enzyme with a distinct substrate binding pocket. These structures demonstrate how chiral discrimination by R- and S-HPCDH results from alternative binding of the distal end of substrates within each substrate binding pocket.
Asunto(s)
Mesna/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estereoisomerismo , Especificidad por Sustrato , Xanthobacter/enzimologíaRESUMEN
(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 S-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, 1315) 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 a larger contributor to S-HPC binding to S-HPCDH than for R-HPC binding to R-HPCDH. It is proposed that active site constraints within the S-HPCDH prevent proper binding of R-HPC and M-HPC due to steric clashes with the improperly aligned methyl group on the C2 carbon, resulting in a different mechanism for controlling substrate specificity and enantioselectivity than present in the R-HPCDH.