Purification and Characterization of a Novel Alcohol Dehydrogenase from Leifsonia sp. Strain S749: a Promising Biocatalyst for an Asymmetric Hydrogen Transfer Bioreduction

To find microorganisms that could reduce phenyl trifluoromethyl ketone (PTK) to (S)-1-phenyltrifluoroethanol [(S)-PTE], styrene-assimilating bacteria (ca. 900 strains) isolated from soil samples were screened. We found that Leifsonia sp. strain S749 was the most suitable strain for the conversion of PTK to (S)-PTE in the presence of 2-propanol as a hydrogen donor. The enzyme corresponding to the reaction was purified homogeneity, characterized and designated Leifsonia alcohol dehydrogenase (LSADH). The purified enzyme had a molecular weight of 110,000 and was composed of four identical subunits (molecular weight, 26,000). LSADH required NADH as a cofactor, showed little activity with NADPH, and reduced a wide variety of aldehydes and ketones. LSADH catalyzed the enantioselective reduction of some ketones with high enantiomeric excesses (e.e.): PTK to (S)-PTE (>99% e.e.), acetophenone to (R)-1-phenylethanol (99% e.e.), and 2-heptanone to (R)-2-heptanol (>99% e.e.) in the presence of 2-propanol without an additional NADH regeneration system. Therefore, it would be a useful biocatalyst.     

Gene cloning and expression of Leifsonia alcohol dehydrogenase (LSADH) involved in asymmetric hydrogen-transfer bioreduction to produce (R)-form chiral alcohols

The gene encoding Leifsonia alcohol dehydrogenase (LSADH), a useful biocatalyst for producing (R)-chiral alcohols, was cloned from the genomic DNA of Leifsonia sp. S749. The gene contained an opening reading frame consisting of 756 nucleotides corresponding to 251 amino acid residues. The subunit molecular weight was calculated to be 24,999, which was consistent with that determined by polyacrylamide gel electrophoresis. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by three column chromatographies. The predicted amino acid sequence displayed 30-50% homology to known short chain alcohol dehydrogenase/reductases (SDRs); moreover, the NADH-binding site and the three catalytic residues in SDRs were conserved. The recombinant E. coli cells which overexpressed lsadh produced (R)-form chiral alcohols from ketones using 2-propanol as a hydrogen donor with the highest level of productivity ever reported and enantiomeric excess (e.e.).     

Chiral alcohol production by NADH-dependent phenylacetaldehyde reductase coupled with in situ regeneration of NADH.

Phenylacetaldehyde reductase (PAR) produced by styrene-assimilating Corynebacterium strain ST-10 was used to synthesize chiral alcohols. This enzyme with a broad substrate range reduced various prochiral aromatic ketones and beta-ketoesters to yield optically active secondary alcohols with an enantiomeric purity of more than 98% enantiomeric excess (e.e.). The Escherichia coli recombinant cells which expressed the par gene could efficiently produce important pharmaceutical intermediates; (R)-2-chloro-1-(3-chlorophenyl)ethanol (28 mg.mL-1) from m-chlorophenacyl chloride, ethyl (R)-4-chloro-3-hydroxy butanoate) (28 mg.mL-1) from ethyl 4-chloro-3-oxobutanoate and (S)-N-tert-butoxycarbonyl(Boc)-3-pyrrolidinol from N-Boc-3-pyrrolidinone (51 mg.mL-1), with more than 86% yields. The high yields were due to the fact that PAR could concomitantly reproduce NADH in the presence of 3-7% (v/v) 2-propanol in the reaction mixture. This biocatalytic process provided one of the best asymmetric reductions ever reported.     

Stereochemical diversity in lignan biosynthesis of Arctium lappa L

The stereochemistry of lignan biosynthesis in Arctium lappa L. is regulated organ-specifically. (+)-Secoisolariciresinol [81% enantiomeric excess (e.e.)] was isolated from A. lappa petioles. In sharp contrast, lignans whose predominant enantiomers have the opposite absolute configuration to that of (+)-secoisolariciresinol [i.e., (-)-matairesinol (>99% e.e.), (-)-arctigenin (>99% e.e.), and (-)-secoisolariciresinol (65% e.e.)] were isolated from seeds of the species. The stereochemical diversity of secoisolariciresinol was demonstrated with enzyme preparations from A. lappa petioles and seeds. Thus, a petiole enzyme preparation catalyzed the formation of (+)-pinoresinol (33% e.e.), (+)-lariciresinol (30% e.e.), and (+)-secoisolariciresinol (20% e.e.) from achiral coniferyl alcohol in the presence of NADPH and H202, whereas that from ripening seeds catalyzed the formation of (-)-pinoresinol (22% e.e.), (-)-lariciresinol (>99% e.e.), and (-)-secoisolariciresinol (38% e.e.) under the same conditions. In addition, the ripening seed enzyme preparation mediated the selective formation of the optically pure (>99% e.e.) (-)-enantiomer of matairesinol from racemic (+/-)-secoisolariciresinols in the presence of NADP. These results indicate that the stereochemical mechanism for lignan biosynthesis in A. lappa varies with organs, suggesting that multiple lignan-synthesizing isozymes are involved in the stereochemical control of lignan formation in A. lappa.     

Preparation of (S)-2,3-dichloro-1-propanol byPseudomonas sp. and its use in the synthesis of (R)-epichlorohydrin

Summary Pseudomonas sp. OS-K-29 assimilated (R)-2,3-dichloro-1-propanol preferentially as the sole source of carbon. Isolation of optically pure (S)-2,3-dichloro-1-propanol with 100% enantiomer excess (e.e.) from the racemate was done based on this bacterial assimilation using immobilized-cells of OS-K-29 with calcium-alginate. The overall examination of the reactor involved 19 batches for 50 days without loss of its activity. Highly pure (R)-epichlorohydrin with 99.5% e.e. was prepared from the (S)-2,3-dichloro-1-propanol with treatment of aqueous NaOH. This new method is simple and useful for manufacturing optically active (S)-2,3-dichloro-1-propanol and (R)-epichlorohydrin.     

Highly efficient synthesis of chiral alcohols with a novel NADH-dependent reductase from Streptomyces coelicolor

An NADH-dependent reductase (ScCR) from Streptomyces coelicolor was discovered by genome mining for carbonyl reductases. ScCR was overexpressed in Escherichia coli BL21, purified to homogeneity and its catalytic properties were studied. This enzyme catalyzed the asymmetric reduction of a broad range of prochiral ketones including aryl ketones, α- and β-ketoesters, with high activity and excellent enantioselectivity (>99% ee) towards β-ketoesters. Among them, ethyl 4-chloro-3-oxobutanoate (COBE) was efficiently converted to ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), an important pharmaceutical intermediate, in water/toluene biphasic system. As much as 600 g/L (3.6 M) of COBE was asymmetrically reduced within 22 h using 2-propanol as a co-substrate for NADH regeneration, resulting in a yield of 93%, an enantioselectivity of >99% ee, and a total turnover number (TTN) of 12,100. These results indicate the potential of ScCR for the industrial production of valuable chiral alcohols.     

Synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate with recombinant Escherichia coli cells expressing (S)-specific secondary alcohol dehydrogenase

The synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-ECHB) from ethyl 4-chloroacetoacetate was studied using whole recombinant cells of Escherichia coli expressing a secondary alcohol dehydrogenase of Candida parapsilosis. Using 2-propanol as an energy source to regenerate NADH, the yield of (R)-ECHB reached 36.6 g/l (more than 99% ee, 95.2% conversion yield) without addition of NADH to the reaction mixture.     

Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus

The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adh(Tt)) was heterologously overexpressed in Escherichia coli, and the protein (ADH(Tt)) was purified to homogeneity and characterized. ADH(Tt) is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to approximately 73 degrees C and a 30-min half-inactivation temperature of approximately 90 degrees C, as well as good tolerance to common organic solvents. ADH(Tt) has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and alpha-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, alpha-tetralone, and alpha-methyl and alpha-ethyl benzoylformates to (S)-(-)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-alpha-(trifluoromethyl)benzyl alcohol (93% ee), (S)-alpha-tetralol (>99% ee), methyl (R)-(-)-mandelate (92% ee), and ethyl (R)-(-)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.     

Continuous production of chiral 1,3-butanediol using immobilized biocatalysts in a packed bed reactor: promising biocatalysis method with an asymmetric hydrogen-transfer bioreduction

An asymmetric hydrogen-transfer biocatalyst consisting of mutated Rhodococcus phenylacetaldehyde reductase (PAR) or Leifsonia alcohol dehydrogenase (LSADH) was applied for some water-soluble ketone substrates. Among them, 4-hydroxy-2-butanone was reduced to (S)/(R)-1,3-butanediol, a useful intermediate for pharmaceuticals, with a high yield and stereoselectivity. Intact Escherichia coli cells overexpressing mutated PAR (Sar268) or LSADH were directly immobilized with polyethyleneimine or 1,6-diaminehexane and glutaraldehyde and evaluated in a batch reaction. This system produced (S)-1,3-butanediol [87% enantiomeric excess (e.e.)] with a space time yield (STY) of 12.5 mg h⁻¹ ml⁻¹ catalyst or (R)-1,3-butanediol (99% e.e.) with an STY of 60.3 mg h⁻¹ ml⁻¹ catalyst, respectively. The immobilized cells in a packed bed reactor continuously produced (R)-1,3-butanediol with a yield of 99% (about 49.5 g/l) from 5% (w/v) 4-hydroxy-2-butanoate over 500 h.     

Purification and characterization of phenylacetaldehyde reductase from a styrene-assimilating Corynebacterium strain, ST-10.

A novel phenylacetaldehyde reductase was purified about 50-fold to homogeneity from Corynebacterium sp. strain ST-10, which can assimilate gaseous styrene as the sole carbon and energy source. The enzyme was inductively synthesized when grown on gaseous styrene and had an important role in styrene metabolism in vivo. The enzyme had a molecular weight of 155,000 and was composed of four identical subunits (molecular weight, 42,000). The enzyme catalyzed the reduction of not only phenylacetaldehyde but also various aldehydes and ketones; however, it did not catalyze the reverse reaction, the dehydrogenation of 2-phenylethanol. The enzyme required NADH as a cofactor and showed no activity with NADPH; therefore, it was defined as an NADH-dependent phenylacetaldehyde reductase. The enzyme stereospecifically produced (S)-(-)-1-phenylethanol from acetophenone; therefore, it would be useful as a biocatalyst.     

NADH peroxidase activity of rubrerythrin

P. S. Alban et al. (J. Appl. Microbiol. (1998) 85, 875-882) reported that a mutant H2O2-resistant strain of Spirullum (S.) volutans showed constitutive overexpression of a protein whose amino acid sequence and molecular weight closely resembled that of a subunit of rubrerythrin, a non-heme iron protein with no known function. They also reported that the mutant strain, but not the wild-type, showed NADH peroxidase activity. Here we demonstrate that rubrerythrin and nigerythrin from Desulfovibrio vulgaris and rubrerythrin from Clostridium perfringens show NADH peroxidase activities in an in vitro system containing NADH, hydrogen peroxide, and a bacterial NADH oxidoreductase. The peroxidase specific activities of the rubrerythrins with the "classical" heme peroxidase substrate, o-dianisidine, are many orders of magnitude lower than that of horseradish peroxidase. These results are consistent with the phenotype of the H2O2-resistant strain of S. volutans. The reaction of reduced (i.e., all-ferrous) rubrerythrin with excess O2 takes several minutes, whereas the anaerobic reaction of reduced rubrerythrin with hydrogen peroxide is on the millisecond time scale and results in full oxidation of all iron centers to their ferric states. Rubrerythrins could, thus, function as the terminal components of NADH peroxidases in air-sensitive bacteria and archaea.     

Microbial production of methyl ketones. Purification and properties of a secondary alcohol dehydrogenase from yeast.

Cell-free extracts derived from yeasts Candida utilis ATCC 26387, Hansenula polymorpha ATCC 26012, Pichia sp. NRRL-Y-11328 Torulopsis sp. strain A1 and Kloeckera sp. strain A2 catalyzed an NAD+-dependent oxidation of secondary alcohols (2-propanol, 2-butanol, 2-pentanol, 2-hexanol) to the corresponding methyl ketones (acetone, 2-butanone, 2-pentanone, 2-hexanone). We have purified a NAD+-specific secondary alcohol dehydrogenase from methanol-grown yeast, Pichia sp. The purified enzyme is homogenous as judged by polyacrylamide gel electrophoresis. The purified enzyme catalyzed the oxidation of secondary alcohols to the corresponding methyl ketones in the presence of NAD+ as an electron acceptor. Primary alcohols were not oxidized by the purified enzyme. The optimum pH for oxidation of secondary alcohols by the purified enzyme is 8.0. The molecular weight of the purified enzyme as determined by gel filtration is 98 000 and subunit size as determined by sodium dodecyl sulfate gel electrophoresis is 48 000. The activity of the purified secondary alcohol dehydrogenase was inhibited by sulfhydryl inhibitors and metal-binding agents.     

Biochemical characterization and substrate profiling of a new NADH-dependent enoate reductase from Lactobacillus casei

Carbon-carbon double bond of α,β-unsaturated carbonyl compounds can be reduced by enoate reductase (ER), which is an important reaction in fine chemical synthesis. A putative enoate reductase gene from Lactobacillus casei str. Zhang was cloned into pET-21a+ and expressed in Escherichia coli BL21 (DE3) host cells. The encoded enzyme (LacER) was purified by ammonium sulfate precipitation and treatment in an acidic buffer. This enzyme was identified as a NADH-dependent enoate reductase, which had a K(m) of 0.034 ± 0.006 mM and k(cat) of (3.2 ± 0.2) × 103 s?1 toward NADH using 2-cyclohexen-1-one as the substrate. Its K(m) and k(cat) toward substrate 2-cyclohexen-1-one were 1.94 ± 0.04 mM and (8.4 ± 0.2) × 103 s?1, respectively. The enzyme showed a maximum activity at pH 8.0-9.0. The optimum temperature of the enzyme was 50-55°C, and LacER was relatively stable below 60 °C. The enzyme was active toward aliphatic alkenyl aldehyde, ketones and some cyclic anhydrides. Substituted groups of cyclic α,β-unsaturated ketones and its ring size have positive or negative effects on activity. (R)-(-)-Carvone was reduced to (2R,5R)-dihydrocarvone with 99% conversion and 98% (diasteromeric excess: de) stereoselectivity, indicating a high synthetic potential of LacER in asymmetric synthesis.     

Benzylic alcohols as stereospecific substrates and inhibitors for aryl sulfotransferase

Aryl sulfotransferase IV catalyzes the 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent formation of sulfuric acid esters of benzylic alcohols. Since the benzylic carbon bearing the hydroxyl group can be asymmetric, the possibility of stereochemical control of substrate specificity of the sulfotransferase was investigated with benzylic alcohols. Benzylic alcohols of known stereochemistry were examined as potential substrates and inhibitors for the homogeneous enzyme purified from rat liver. For 1-phenylethanol, both the (+)-(R)- and (-)-(S)-enantiomers were substrates for the enzyme, and the kcat/Km value for the (-)-(S)-enantiomer was twice that of the (+)-(R)-enantiomer. The enzyme displayed an absolute stereospecificity with ephedrine and pseudoephedrine, and with 2-methyl-1-phenyl-1-propanol; that is, only (-)-(1R,2S)-ephedrine, (-)-(1R,2R)-pseudoephedrine, and (-)-(S)-2-methyl-1-phenyl-1-propanol were substrates for the sulfotransferase. In the case of 1,2,3,4-tetrahydro-1-naphthol, only the (-)-(R)-enantiomer was a substrate for the enzyme. Both (+)-(R)-2-methyl-1-phenyl-1-propanol and (+)-(S)-1,2,3,4-tetrahydro-1-naphthol were competitive inhibitors of the aryl sulfotransferase-catalyzed sulfation of 1-naphthalenemethanol. Thus, the configuration of the benzylic carbon bearing the hydroxyl group determined whether these benzylic alcohols were substrates or inhibitors of the rat hepatic aryl sulfotransferase IV. Furthermore, benzylic alcohols such as (+)-(S)-1,2,3,4-tetrahydro-1-naphthol represent a new class of inhibitors for the aryl sulfotransferase.     

Cloning and expression of the gene encoding (R)-specific carbonyl reductase from Candida parapsilosis CCTCC M203011

The gene which encodes (R)-specific carbonyl reductase (rCR) from Candida parapsilosis CCTCC M203011 was cloned, sequenced and compared with genes from the GenBank. The results indicated that rCR gene was 1011 bp, encoding a protein of 336 amino acids with a molecular weight of 35.9 kDa, and its nucleotide sequence showed 99% similarity to those of other members of the alcohol dehydrogenase superfamily. The rCR gene could express in recombinant strain Escherichia coli JM 109, and the expression plasmid could produce (R)-1-pheny-1,2-ethanediol (100% e.e., 80.14% yield) fromβ-hydroxyacetophenone without any additive to regenerate NAD+ from NADH.     

A genomic search approach to identify carbonyl reductases in Gluconobacter oxydans for enantioselective reduction of ketones

The versatile carbonyl reductases from Gluconobacter oxydans in the enantioselective reduction of ketones to the corresponding alcohols were exploited by genome search approach. All purified enzymes showed activities toward the tested ketoesters with different activities. In the reduction of 4-phenyl-2-butanone with in situ NAD(P)H regeneration system, (S)-alcohol was obtained with an e.e. of up to 100% catalyzed by Gox0644. Under the same experimental condition, all enzymes catalyzed ethyl 4-chloroacetoacetate to give chiral products with an excellent e.e. of up to 99%, except Gox0644. Gox2036 had a strict requirement for NADH as the cofactor and showed excellent enantiospecificity in the synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate. For the reduction of ethyl 2-oxo-4-phenylbutyrate, excellent e.e. (>99%) and high conversion (93.1%) were obtained by Gox0525, whereas the other enzymes showed relatively lower e.e. and conversions. Among them, Gox2036 and Gox0525 showed potentials in the synthesis of chiral alcohols as useful biocatalysts.     

Purification and characterization of an anti-Prelog alcohol dehydrogenase from <Emphasis Type="Italic">Oenococcus oeni</Emphasis> that reduces 2-octanone to (<Emphasis Type="Italic">R</Emphasis>)-2-octanol

An anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol was purified by 26-fold to homogeneity. The enzyme had a homodimeric structure consisting of 49 kDa subunits, required NADPH, but not NADH, as a cofactor and was a Zn-independent short-chain dehydrogenase. Aliphatic methyl ketones (chain length ≥6 carbon atoms) and aromatic methyl ketones were the preferred substrates for the enzyme, the best being 2-octanone. Maximum enzyme activity with 2-octanone was at 45°C and at pH 8.0.     

Oxidation of naphthalene by a multicomponent enzyme system from Pseudomonas sp. strain NCIB 9816.

The initial reactions in the oxidation of naphthalene by Pseudomonas sp. strain NCIB 9816 involves the enzymatic incorporation of one molecule of oxygen into the aromatic nucleus to form (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. The enzyme catalyzing this reaction, naphthalene dioxygenase, was resolved into three protein components, designated A, B, and C, by DEAE-cellulose chromatography. Incubation of naphthalene with components A, B, and C in the presence of NADH resulted in the formation of (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. The ratio of oxygen and NADH utilization to product formation was 1:1:1. NADPH also served as an electron donor for naphthalene oxygenation. However, its activity was less than 50% of that observed with NADH. Component A showed NAD(P)H-cytochrome c reductase activity which was stimulated by the addition of flavin adenine dinucleotide and flavin mononucleotide. A similar stimulation was observed when these flavin nucleotides were added to the naphthalene dioxygenase assay system. These preliminary observations indicate that naphthalene dioxygenase has properties in common with both monooxygenase and dioxygenase multicomponent enzyme systems.     

A new alcohol dehydrogenase with unique stereospecificity from Pseudomonas sp.

A new nicotinamide cofactor-dependent alcohol dehydrogenase from Pseudomonas strain SBD6 (PADH) was isolated and purified 150-fold to homogeneity using a combination of salt precipitation, anion-exchange chromatography, gel filtration chromatography, and dye matrix chromatography. Approximately 10 mg of pure enzyme can be obtained from 10 g of wet cells. The enzyme has four subunits with a total molecular weight of 162,000. Incubation with the metal chelators 1,10-phenanthroline, 2-aminoethanethiol, hydroxyquinolinesulfonic acid, N-ethylmaleimide, and potassium cyanide result in complete loss of activity. The enzyme is very stable (t_1/2 7 days at pH 7 and 25°C in the absence of 2-propanol and 18 days in the presence of 10% 2-propanol, v/v) and possesses a broad substrate specificity with transfer of the pro-(R) hydride from NADH to the si face of carbonyl substrates to give (R)-alcohols in high enantiomeric excess, a stereochemical process different from that of other known alcohol dehydrogenases. Synthetic scale reductions are facilitated with 2-propanol as a hydride source for the regeneration of NADH. The kinetic mechanism is ordered bi-bi with the cofactor binding first. Based on NAD and 2-propanol, the kinetic parameters of the enzyme were determined to be V_max = 29.9 Units mg⁻¹ at 25°C and pH 8.5, K_m^NAD = 0.36 m and K_m^2-propanol = 0.19 m .     

The stereoselectivity and catalytic properties of Xanthobacter autotrophicus 2-[(R)-2-Hydroxypropylthio]ethanesulfonate dehydrogenase are controlled by interactions between C-terminal arginine residues and the sulfonate of coenzyme M

2-[(R)-2-Hydroxypropylthio]ethanesulfonate (R-HPC) dehydrogenase (DH) catalyzes the reversible oxidation of R-HPC to 2-(2-ketopropylthio)ethanesulfonate (2-KPC) in a key reaction in the bacterial conversion of chiral epoxides to beta-keto acids. R-HPCDH is highly specific for the R-enantiomer of HPC, while a separate enzyme, S-HPCDH, catalyzes the oxidation of the corresponding S-enantiomer. In the present study, the features of substrate and enzyme imparting stereospecificity have been investigated for R-HPCDH. S-HPC was a substrate for R-HPCDH with a K(m) identical to that for R-HPC but with a k(cat) 600 times lower. Achiral 2-propanol and short-chain (R)- and (S)-2-alkanols were substrates for R-HPCDH. For (R)-alkanols, as the carbon chain length increased, K(m) decreased, with the K(m) for (R)-2-octanol being 1700 times lower than for 2-propanol. At the same time, k(cat) changed very little and was at least 90% lower than k(cat) for R-HPC and at least 22 times higher than k(cat) for S-HPC. (S)-2-Butanol and (S)-2-pentanol were substrates for R-HPCDH. The K(m) for (S)-2-butanol was identical to that for (R)-2-butanol, while the K(m) for (S)-2-pentanol was 7.5 times higher than for (R)-2-pentanol. Longer chain (S)-2-alkanols were sufficiently poor substrates for R-HPCDH that kinetic parameters could not be determined. Mutagenesis of C-terminal arginine residues of R-HPCDH revealed that R152 and R196 are essential for effective catalysis with the natural substrates R-HPC and 2-KPC but not for catalysis with 2-alkanols or ketones as substrates. Short-chain alkylsulfonates and coenzyme M (2-mercaptoethanesulfonate) were found to modify the kinetic parameters for 2-butanone reduction by R-HPCDH in a saturable fashion, with the general effect of increasing k(cat), decreasing K(m), and increasing the enantioselectivity of 2-butanone reduction to a theoretical value of 100% (S)-2-butanol. The modulating effects of ethanesulfonate and propanesulfonate provided thermodynamic binding constants close to K(m) for the natural substrates R-HPC and 2-KPC. The effects of alkylsulfonates on modulating the enantioselectivity and kinetic properties of R-HPCDH were abolished in R152A and R196A mutants but not in mutants of other C-terminal arginine residues. Collectively, the results suggest that interactions between the sulfonate of CoM and specific arginine residues are key to the enantioselectivity and catalytic efficiency of R-HPCDH. A model is proposed wherein sulfonate-arginine interactions within an alkylsulfonate binding pocket control the catalytic properties of R-HPCDH.