Publications

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

  Allen J.R., Clark D.D., Krum J.G., Ensign S.A.

  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, coenz ... >> More

  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. << Less

  Proc. Natl. Acad. Sci. U.S.A. 96:8432-8437(1999) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Epoxyalkane: coenzyme M transferase in the ethene and vinyl chloride biodegradation pathways of mycobacterium strain JS60.

  Coleman N.V., Spain J.C.

  Mycobacterium strains that grow on ethene and vinyl chloride (VC) are widely distributed in the environment and are potentially useful for biocatalysis and bioremediation. The catabolic pathway of alkene assimilation in mycobacteria is not well characterized. It is clear that the initial step is a ... >> More

  Mycobacterium strains that grow on ethene and vinyl chloride (VC) are widely distributed in the environment and are potentially useful for biocatalysis and bioremediation. The catabolic pathway of alkene assimilation in mycobacteria is not well characterized. It is clear that the initial step is a monooxygenase-mediated epoxidation that produces epoxyethane from ethene and chlorooxirane from VC, but the enzymes involved in subsequent transformation of the epoxides have not been identified. We investigated epoxyethane metabolism in Mycobacterium strain JS60 and discovered a coenzyme M (CoM)-dependent enzyme activity in extracts from VC- and ethene-grown cells. PCR amplifications using primers targeted at epoxyalkane:CoM transferase (EaCoMT) genes yielded part of the JS60 EaCoMT gene, which was used to clone an 8.4-kb genomic DNA fragment. The complete EaCoMT gene (etnE) was recovered, along with genes (etnABCD) encoding a four-component monooxygenase and two genes possibly involved in acyl-CoA ester metabolism. Reverse transcription-PCR indicated that the etnE and etnA genes were cotranscribed and inducible by ethene and VC. Heterologous expression of the etnE gene in Mycobacterium smegmatis mc(2)155 using the pMV261 vector gave a recombinant strain capable of transforming epoxyethane, epoxypropane, and chlorooxirane. A metabolite identified by mass spectrometry as 2-hydroxyethyl-CoM was produced from epoxyethane. The results indicate that the EaCoMT and monooxygenase enzymes encoded by a single operon (etnEABCD) catalyze the initial reactions in both the VC and ethene assimilation pathways. CoM-mediated reactions appear to be more widespread in bacteria than was previously believed. << Less

  J Bacteriol 185:5536-5545(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Kinetic and microcalorimetric analysis of substrate and cofactor interactions in epoxyalkane:CoM transferase, a zinc-dependent epoxidase.

  Krum J.G., Ellsworth H., Sargeant R.R., Rich G., Ensign S.A.

  Epoxyalkane:CoM transferase (EaCoMT) is a key enzyme of bacterial propylene metabolism, catalyzing the nucleophilic attack of coenzyme M (CoM, 2-mercaptoethanesulfonic acid) on epoxypropane to form the thioether conjugate 2-hydroxypropyl-CoM. The biochemical and molecular properties of EaCoMT sugg ... >> More

  Epoxyalkane:CoM transferase (EaCoMT) is a key enzyme of bacterial propylene metabolism, catalyzing the nucleophilic attack of coenzyme M (CoM, 2-mercaptoethanesulfonic acid) on epoxypropane to form the thioether conjugate 2-hydroxypropyl-CoM. The biochemical and molecular properties of EaCoMT suggest that the enzyme belongs to the family of alkyltransferase enzymes for which Zn plays a key role in activating an organic thiol substrate for nucleophilic attack on an alkyl-donating substrate. In the present work, the role of Zn in the EaCoMT-catalyzed reactions is established by removing Zn from EaCoMT, resulting in loss of catalytic activity that was restored upon addition of Zn back to the enzyme, and by expressing an inactive and Zn-deficient form of the enzyme that was activated by addition of ZnCl(2) or CoCl(2). Site-directed mutagenesis of one of the predicted Zn ligands (C220A) resulted in the formation of a largely catalytically inactive protein (0.06% of wild-type activity) that, when purified, contained a substoichiometric complement of Zn. EaCoMT was kinetically characterized and found to follow a random sequential mechanism with kinetic parameters K(m,epoxypropane) = 1.8 microM, K(m,CoM) = 34 microM, and k(cat) = 6.5 s(-1). The CoM analogues 2-mercaptopropionate, 2-mercaptoethanol, and cysteine substituted poorly for CoM as the thiol substrate, with specific rates of epoxyalkane conjugation that were at best 0.6% of the CoM-dependent rate, while ethanethiol, propanethiol, glutathione, homocysteine, and lipoic acid provided no activity. 2-Mercaptoethanol was a weak competitive inhibitor vs CoM with a K(I) of 192 mM. Isothermal titration calorimetry was used to investigate the thermodynamic binding determinants for the interaction of CoM and analogues with holo, Zn-deficient, and C220A EaCoMT variants. The stoichiometry of CoM binding correlated directly with the Zn content rather than monomer content of protein samples, reinforcing the importance of Zn in CoM binding. The binding of CoM to EaCoMT occurred with DeltaG = -7.5 kcal/mol (K(d) = 3.8 microM) and was driven by a large release of enthalpy. The thermodynamic contributors (K(a), DeltaG, DeltaH, DeltaS) to the individual binding of CoM, ethanesulfonate, and ethanethiol were determined and used to assess the contributions of the thiol, alkyl, and sulfonate moieties to total binding energy in the E x CoM binary complex. << Less

  Biochemistry 41:5005-5014(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.