Structural Basis for Stereoselectivity in the (R)- and (S)-Hydroxypropylthioethanesulfonate Dehydrogenases †,‡ Arathi M. Krishnakumar, § Boguslaw P. Nocek, § Daniel D. Clark, | Scott A. Ensign, | and John W. Peters* ,§ Department of Chemistry and Biochemistry, Montana State UniVersity, Bozeman, Montana 59717, and Department of Chemistry and Biochemistry, Utah State UniVersity, Logan, Utah 84322 ReceiVed February 20, 2006; ReVised Manuscript ReceiVed May 22, 2006 ABSTRACT: 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 Å 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. The highly reactive nature of epoxides makes them valuable intermediates for the synthesis of more complex optically active or biologically active compounds, including pharmaceuticals and agrichemicals. For this reason, there has been considerable interest in exploiting epoxide-utilizing enzymes which have stereoselective and regioselective prop- erties for producing enantiopure compounds. Some of the known alkene-oxidizing enzymes, such as heme-dependent monooxygenases, ω-hydroxylases, and non-heme oxygena- ses, catalyze direct stereospecific epoxidation of alkenes (1). Alternatively, enantiopure epoxides can be resolved from racemic mixtures by enantioselective epoxide metabolizing enzymes. Some microorganisms are capable of utilizing aliphatic alkenes and epoxides as sole carbon sources (2). The pathway for the metabolism of short chain alkenes and epoxides in aerobic bacterium Xanthobacter autotrophicus Py2 has been well-characterized (3-8). Integral to this metabolic pathway are the two stereospecific 2-hydroxypro- pylthioethanesulfonate dehydrogenases designated R-HPCDH 1 and S-HPCDH that individually allow for the oxidation of only (R)- and (S)-epoxypropane enantiomers, respectively, to the common achiral product 2-ketopropylthioethane- sulfonate or 2-ketopropyl-CoM. The two dehydrogenases, R-HPCDH and S-HPCDH, are related with 41% similar sequences and belong to the short chain dehydrogenase reductase superfamily of enzymes (8). Enzymes of this class are approximately 250 amino acids in † This work was supported by Department of Energy Grant DE- FG02-04ER15563 (to J.W.P.) and National Institutes of Health Grant GM51805 (to S.A.E.). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), a national user facility operated by Stanford University on behalf of the U.S. Depart- ment of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedi- cal Technology Program, and the National Institute of General Medical Sciences. ‡ Coordinates for the R-HPCDH-2-KPC cocrystal structure have been deposited in the Protein Data Bank as entry 2cfc. * To whom correspondence should be addressed. Phone: (406) 994- 7211. Fax: (406) 994-7212. E-mail: john.peters@chemistry.montana.edu. § Montana State University. | Utah State University. 1 Abbreviations: R-HPC, 2-(R)-hydroxypropylthioethanesulfonate; S-HPC, 2-(S)-hydroxypropylthioethanesulfonate; R-HPCDH, 2-(R)- hydroxypropylthioethanesulfonate dehydrogenase; S-HPCDH, 2-(S)- hydroxypropylthioethanesulfonate dehydrogenase; SDR, short chain dehydrogenase reductase; 2-KPC, 2 ketopropylthioethanesulfonate; CoM, coenzyme M (2-mercaptoethanesulfonate); 2-KPCC, 2-ketopro- pyl-CoM oxidoreductase/carboxylase; MAD, multiple-wavelength anoma- lous diffraction; NAD + and NADH, nicotinamide adenine dinucleotide; NADP + , nicotinamide adenine dinucleotide phosphate; MDH, mannitol dehydrogenase; HADH, halo alcohol dehydrogenase. 8831 Biochemistry 2006, 45, 8831-8840 10.1021/bi0603569 CCC: $33.50 © 2006 American Chemical Society Published on Web 06/30/2006