A role for coenzyme M (2-mercaptoethanesulfonic acid) in a bacterial pathway of aliphatic epoxide carboxylation

Abstract

The bacterial metabolism of short-chain aliphatic alkenes occurs via oxidation to epoxyalkanes followed by carboxylation to beta-ketoacids. Epoxyalkane carboxylation requires four enzymes (components I-IV), NADPH, NAD(+), and a previously unidentified nucleophilic thiol. In the present work, coenzyme M (2-mercaptoethanesulfonic acid), a compound previously found only in the methanogenic Archaea where it serves as a methyl group carrier and activator, has been identified as the thiol and central cofactor of aliphatic epoxide carboxylation in the Gram-negative bacterium Xanthobacter strain Py2. Component I catalyzed the addition of coenzyme M to epoxypropane to form a beta-hydroxythioether, 2-(2-hydroxypropylthio)ethanesulfonate. Components III and IV catalyzed the NAD(+)-dependent stereoselective dehydrogenation of R- and S-enantiomers of 2-(2-hydroxypropylthio)ethanesulfonate to form 2-(2-ketopropylthio)ethanesulfonate. Component II catalyzed the NADPH-dependent cleavage and carboxylation of the beta-ketothioether to form acetoacetate and coenzyme M. These findings evince a newfound versatility for coenzyme M as a carrier and activator of alkyl groups longer in chain-length than methane, a function for coenzyme M in a catabolic pathway of hydrocarbon oxidation, and the presence of coenzyme M in the bacterial domain of the phylogenetic tree. These results serve to unify bacterial and Archaeal metabolism further and showcase diverse biological functions for an elegantly simple organic molecule.

Figure 1

Stimulation of epoxide carboxylase activity by commercially obtained CoM. Assays were performed in duplicate in sealed 9-ml vials containing purified components (5 μg of component I, 250 μg of component II, 40 μg of component III, and 25 μg of component IV) in 50 mM Tris⋅HCl (pH 8.2). □, assays containing as-isolated component I; ○, assays containing component I preincubated with epoxypropane followed by gel filtration chromatography.

Figure 2

Spectral identification of epoxypropane-thiol adduct. (A) ¹H NMR spectrum of the epoxypropane–cofactor adduct isolated from component I. (B) ¹H NMR spectrum of the product of component I-catalyzed reaction of CoM with epoxypropane. (C) ¹H NMR spectrum of chemically synthesized 2-hydroxypropyl–CoM. There are five signals (a–e) that correspond to protons on the carbon atoms as indicated in C. The triplet resonances at 2.91 and 3.16 ppm correspond to methylene groups (a) and (b), each integrating to two protons. The protons of methylene group (c) are not chemically equivalent and are therefore split into two quartets with resonances centered at 2.64 and 2.75 ppm, each multiplet integrating to one proton. The sextet at 3.98 ppm corresponds to the proton on carbon (d) and integrates to one proton. The protons of methyl group (e) are split to a doublet at 1.23 ppm that integrates to three protons. The resonance at 1.89 ppm in the isolated epoxypropane–cofactor adduct spectrum (A) is caused by acetate remaining in the sample.

Figure 3

¹H NMR spectrum of the product of component III- and IV-catalyzed reaction of NAD⁺ and 2-hydroxypropyl–CoM. The spectra of chemically synthesized 2-hydroxypropyl–CoM (in blue) and 2-ketopropyl–CoM (in red) are overlaid and shown directly above the product spectrum. Resonances at 1.23 and 3.98 ppm correspond to methyl group (e) and proton (d) of 2-hydroxypropyl–CoM, respectively. Resonances at 2.63 and 2.74 ppm correspond to methylene group (c) of 2-hydroxypropyl–CoM. The multiplets at 2.89 and 3.15 ppm are the overlapping resonances of methylene groups (b, b′) and (a, a′) of 2-hydroxypropyl–CoM and 2-ketopropyl–CoM, respectively. The singlet at 3.63 ppm corresponds to the (c′) methylene group of 2-ketopropyl–CoM, which integrates to two protons. The signal at 2.33 ppm corresponds to methyl group (e′) of 2-ketopropyl–CoM, which integrates to three protons.

Figure 4

¹³C NMR spectrum of the product of component II-catalyzed reaction of 2-ketopropyl–CoM, NaH¹³CO₃, and NADPH. The resonance peak at 174.6 ppm corresponds to the chemical shift for the C₁ (carboxyl) atom of acetoacetate as reported (8). The resonance at 160.3 ppm is caused by the NaH¹³CO₃ present in the sample. The resonance at 77.0 ppm is caused by chloroform, which was used as the reference.

Figure 5

Roles of CoM in epoxide carboxylation (A) and methanogenesis (B).