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SUMMARY Coenzyme M (2-mercaptoethanesulfonate; CoM) is one of several atypical cofactors discovered in methanogenic archaea which participate in the biological reduction of CO2 to methane. Elegantly simple, CoM, so named for its role as a methyl carrier in all methanogenic archaea, is the smallest known organic cofactor. It was thought that this cofactor was used exclusively in methanogenesis until it was recently discovered that CoM is a key cofactor in the pathway of propylene metabolism in the gram-negative soil microorganism Xanthobacter autotrophicus Py2. A four-step pathway requiring CoM converts propylene and CO2 to acetoacetate, which feeds into central metabolism. In this process, CoM is used to activate and convert highly electrophilic epoxypropane, formed from propylene epoxidation, into a nucleophilic species that undergoes carboxylation. The unique properties of CoM provide a chemical handle for orienting compounds for site-specific redox chemistry and stereospecific catalysis. The three-dimensional structures of several of the enzymes in the pathway of propylene metabolism in defined states have been determined, providing significant insights into both the enzyme mechanisms and the role of CoM in this pathway. These studies provide the structural basis for understanding the efficacy of CoM as a handle to direct organic substrate transformations at the active sites of enzymes.

Epoxide metabolism in Xanthobacter autotrophicus Py2 results in the conversion of epoxypropane to acetoacetate. Epoxide metabolism is initiated by the nucleophilic addition of coenzyme M to the (R)- and (S)-enantiomers of epoxypropane which forms the respective enantiomers of 2-hydroxypropyl-coenyme M. The (R)- and (S)-enantiomers of 2-hydroxypropyl coenzyme are oxidized to the achiral product 2-ketopropyl-CoM by two stereoselective dehydrogenases. The dehydrogenases catalyzing these reactions, termed (R)-hydroxypropyl-coenzyme M dehydrogenase (R-HPCDH) and (S)-hydroxypropyl-coenzyme M dehydrogenase (S-HPCDH), are NAD(+)-dependent enzymes belonging to the short chain dehydrogenase/reductase (SDR) family of enzymes. In this study, the crystal structure of R-HPCDH cocrystallized in the presence of (S)-hydroxypropyl-coenzyme M has been determined using X-ray diffraction methods and refined to 1.8 A resolution. The structure of R-HPCDH is tetrameric and stabilized by the interaction of the terminal carboxylates of each subunit with divalent metal ions. The structure of the presumed product-bound state reveals that binding interactions between the negatively charged oxygen atoms of the sulfonate moiety have striking similarities to sulfonate interactions observed in the previously determined structure of 2-ketopropyl-CoM oxidoreductase/carboxylase, highlighting the utility of coenzyme M as a carrier molecule in the pathway. The key elements of the aforementioned interactions are electrostatic interactions between the sulfonate oxygen atoms and two arginine residues (R152 and R196) of R-HPCDH. The comparison of the structure of R-HPCDH with a homology model of S-HPCDH provides a structural basis for a mechanism of substrate specificity in which the binding of the substrate sulfonate moiety at distinct sites on each stereoselective enzyme directs the orientation of the appropriate substrate enantiomer for hydride abstraction.

(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...

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