Characterization of 2-Bromoethanesulfonate as a Selective Inhibitor of the Coenzyme M-Dependent Pathway and Enzymes of Bacterial Aliphatic Epoxide Metabolism

Abstract

Bacterial growth with short-chain aliphatic alkenes requires coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which serves as the nucleophile for activation and conversion of epoxide products formed from alkene oxidation to central metabolites. In the present work the CoM analog 2-bromoethanesulfonate (BES) was shown to be a specific inhibitor of propylene-dependent growth of and epoxypropane metabolism by Xanthobacter autotrophicus strain Py2. BES (at low [millimolar] concentrations) completely prevented growth with propylene but had no effect on growth with acetone or n-propanol. Propylene consumption by cells was largely unaffected by the presence of BES, but epoxypropane accumulated in the medium in a time-dependent fashion with BES present. The addition of BES to cells resulted in time-dependent loss of epoxypropane degradation activity that was restored upon removal of BES and addition of CoM. Exposure of cells to BES resulted in a loss of epoxypropane-dependent CO 2 fixation activity that was restored only upon synthesis of new protein. Addition of BES to cell extracts resulted in an irreversible loss of epoxide carboxylase activity that was restored by addition of purified 2-ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of epoxide carboxylation, but not by addition of epoxyalkane:CoM transferase or 2-hydroxypropyl-CoM dehydrogenase, the enzymes which catalyze the first two reactions of epoxide carboxylation. Comparative studies of the propylene-oxidizing actinomycete Rhodococcus rhodochrous strain B276 showed that BES is an inhibitor of propylene-dependent growth in this organism as well but is not an inhibitor of CoM-independent growth with propane. These results suggest that BES inhibits propylene-dependent growth and epoxide metabolism via irreversible inactivation of the key CO 2 -fixing enzyme 2-KPCC.

Mechanism of Inhibition of Aliphatic Epoxide Carboxylation by the Coenzyme M Analog 2-Bromoethanesulfonate

“…The present work expands on our previous work (16) showing BES to be an inactivator of bacterial epoxide metabolism by determining the targets and mechanisms of BES inhibition and inactivation. R-HPCDH and 2-KPCC were reversibly inhibited by BES, whereas 2-KPCC was additionally inactivated by the highly specific and irreversible alkylation of the interchange thiol that interacts with the substrate 2-KPC within the enzyme active site.…”

“…Time-and Concentration-dependent Inactivation of 2-KPCC by BES-A previous study showed that the addition of BES to cell extracts of X. autotrophicus resulted in an irreversible loss of epoxide carboxylation activity that could only be restored by adding purified active 2-KPCC back to the cell extracts, suggesting that 2-KPCC is irreversibly inactivated by BES (16). To investigate this in more detail, 2-KPCC was incubated anoxically in the presence of DTT with various concentrations of BES, and samples were removed at various time points and assayed for activity after removal of BES as described above.…”

“…Treatment of propylene-grown X. autotrophicus with BES resulted in loss of epoxide carboxylation activity that could only be restored by new protein synthesis. The addition of purified 2-KPCC restored epoxide carboxylase activity in cell extracts inactivated with BES, suggesting that 2-KPCC is the target of BES inactivation (16).…”

“…Based on these studies of BES inhibition of methanogens and MCR, BES was studied as a possible inhibitor of bacterial alkene metabolism and found to specifically inhibit growth of Xanthobacter autotrophicus Py2 and Rhodococcus rhodochrous B276 with propylene, but not with carbon sources that do not use the CoM-dependent pathway and enzymes of Fig. 1 (16). Treatment of propylene-grown X. autotrophicus with BES resulted in loss of epoxide carboxylation activity that could only be restored by new protein synthesis.…”

Archaeoglobus infectus sp. nov., a novel thermophilic, chemolithoheterotrophic archaeon isolated from a deep-sea rock collected at Suiyo Seamount, Izu-Bonin Arc, western Pacific Ocean

“…Coenzyme M is an essential terminal methyl carrier in methanogenesis (Graham & White, 2002;Thauer, 1998) and promotes growth in some methanogenic members of the Archaea (Miller et al, 1986;Sprenger et al, 2000;. Coenzyme M has a role not only in the methanogenesis pathway in the Euryarchaeota, but also in the utilization of short-chain alkenes, as demonstrated in recent studies on propylene-, ethyleneand vinyl chloride-utilizing bacteria (Allen et al, 1999;Boyd et al, 2006;Coleman & Spain, 2003a, b;Danko et al, 2006;Mattes et al, 2005). Emission of trace amounts of methane is generally observed in the growing cultures of almost all Archaeoglobus species, but they are unable to grow by methanogenesis.…”