The bacterial metabolism of short-chain aliphatic alkenes occurs via oxidation to epoxyalkanes followed by carboxylation to ␤-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 ␤-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 ␤-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.

Epoxide metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by an NADPH-and NAD ؉ -dependent carboxylation reaction that forms ␤-keto acids as products. Epoxide carboxylase, the enzyme catalyzing this reaction, was resolved from the soluble fraction of cell-free extracts into four protein components that are obligately required for functional reconstitution of epoxide carboxylase activity. One of these components, component II, has previously been purified and characterized as an NADPH:disulfide oxidoreductase. In the present study, the three additional epoxide carboxylase components have been purified to homogeneity and characterized. Xanthobacter strain Py2 is one of several bacteria capable of growth with short chain aliphatic alkenes as carbon and energy sources (1). The first step in alkene metabolism involves an oxidative insertion of an oxygen atom into the olefin bond in a reaction that is catalyzed by an inducible (2), multiprotein (3) alkene monooxygenase as shown for the substrate propylene and product epoxypropane in Equation 1.Epoxides formed in this manner are further metabolized via a novel ring opening and carboxylation reaction that requires CO 2 as a cosubstrate and forms a ␤-keto acid as product as shown in Equation 2 (4, 5).Aliphatic epoxides such as epoxypropane have toxic, mutagenic, and potential carcinogenic properties (6), and their metabolism in bacteria and mammalian systems has been the focus of considerable research in recent years. Epoxide carboxylation as described for Xanthobacter Py2 represents the most recently discovered biological epoxide transformation reaction, the others involving conjugation to glutathione, hydration to dihydrodiols (7), or isomerization to an aldehyde (8). Initial studies of the epoxide-carboxylating enzyme, designated an epoxide carboxylase, indicate that it has cofactor requirements, molecular properties, and a catalytic mechanism as unique as the epoxide carboxylation reaction itself.With respect to cofactor requirements, in vitro epoxide carboxylation requires a source of reductant (DTT, 1 other dithiols, or NADPH) and an oxidant (NAD ϩ ) (4, 9). These cofactor requirements are unprecedented for all other carboxylases that have been characterized. The requirement of oxidant and reductant is intriguing since there is no net redox chemistry involved in epoxide carboxylation. In the course of epoxide carboxylation, there is an apparent transhydrogenation reaction wherein the reductant becomes oxidized and NAD ϩ becomes reduced, although this has not to date been unequivocally demonstrated.With respect to molecular properties, epoxide carboxylase appears to function as a multiprotein complex (10, 11). Fractionation of Xanthobacter cell extracts by anion-exchange chromatography resolved epoxide carboxylase into three fractions, designated fractions I, II, and III based on their order of elution, that could be recombined with restoration of activity (11). The active component of one of these fractions was purified to homogeneity on the basi...

A novel enzymatic reaction involved in the metabolism of aliphatic epoxides by Xanthobacter strain Py2 is described. Cell extracts catalyzed the CO 2 -dependent carboxylation of propylene oxide (epoxypropane) to form acetoacetate and ␤-hydroxybutyrate. The time courses of acetoacetate and ␤-hydroxybutyrate formation indicate that acetoacetate is the primary product of propylene oxide carboxylation and that ␤-hydroxybutyrate is a secondary product formed by the reduction of acetoacetate. Analogous C 5 carboxylation products were identified with 1,2-epoxybutane as the substrate. In the absence of CO 2 , propylene oxide and 1,2-epoxybutane were isomerized to form acetone and methyl ethyl ketone, respectively, as dead-end products. The carboxylation of short-chain epoxides to ␤-keto acids is proposed to serve as the physiological reaction for the metabolism of aliphatic epoxides in Xanthobacter strain Py2.Xanthobacter strain Py2 is one of several bacteria capable of aerobic growth with aliphatic alkenes as carbon and energy sources (4). The pathway of alkene metabolism involves an initial monooxygenase-catalyzed reaction producing epoxide intermediates (12), as shown for the substrate propylene and the product propylene oxide (epoxypropane) in the following equation:Aliphatic epoxides such as propylene oxide have toxic, mutagenic, and potential carcinogenic properties (2, 13), and their metabolism in bacteria and mammalian systems has been the focus of considerable research in recent years. The best-characterized epoxide-degrading enzymes are the detoxifying epoxide hydrolases of mammalian cells, which hydrate epoxides to dihydrodiols (11). The hydrolysis of epoxides to diols has also been described as a bacterial mechanism for the utilization of epoxides as carbon and energy sources (1, 6). Another mechanism has been described for styrene-degrading bacteria, which metabolize the styrene epoxidation product, styrene oxide, via an isomerization reaction which yields phenylacetaldehyde as an intermediate product (5,8).The isomerization of short-chain epoxides to ketones (Fig. 1A) has been observed in whole-cell suspensions (10) and cell extracts of Xanthobacter strain Py2 (14). However, these ketones are not further metabolized, suggesting that they are not the physiological products of epoxide conversions. Recently, we demonstrated that the isomerization of epoxides to ketones by whole-cell suspensions of Xanthobacter Py2 occurred only when CO 2 was excluded from the assay mixture (9). This observation was extended to demonstrate that the metabolism of propylene oxide proceeds by a CO 2 -dependent reaction which was proposed to produce acetoacetate, or a derivative thereof (9). This proposed carboxylation, shown in Fig. 1B, represents a new and novel strategy for epoxide conversion distinct from hydrolytic and isomerization mechanisms such as those described above.To date, no direct evidence for epoxide carboxylation has been provided through in vitro studies. In this study, the in vitro carboxylation of short-chain...

The metabolism of acetone by the aerobic bacterium Xanthobacter strain Py2 was investigated. Cell suspensions of Xanthobacter strain Py2 grown with propylene or glucose as carbon sources were unable to metabolize acetone. The addition of acetone to cultures grown with propylene or glucose resulted in a time-dependent increase in acetone-degrading activity. The degradation of acetone by these cultures was prevented by the addition of rifampin and chloramphenicol, demonstrating that new protein synthesis was required for the induction of acetone-degrading activity. In vivo and in vitro studies of acetone-grown Xanthobacter strain Py2 revealed a CO 2 -dependent pathway of acetone metabolism for this bacterium. The depletion of CO 2 from cultures grown with acetone, but not glucose or n-propanol, prevented bacterial growth. The degradation of acetone by whole-cell suspensions of acetone-grown cells was stimulated by the addition of CO 2 and was prevented by the depletion of CO 2 . The degradation of acetone by acetone-grown cell suspensions supported the fixation of 14 CO 2 into acid-stable products, while the degradation of glucose or ␤-hydroxybutyrate did not. Cultures grown with acetone in a nitrogen-deficient medium supplemented with NaH 13 CO 3 specifically incorporated 13 C-label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-␤-hydroxybutyrate. Cell extracts prepared from acetone-grown cells catalyzed the CO 2 -and ATP-dependent carboxylation of acetone to form acetoacetate as a stoichiometric product. ADP or AMP were incapable of supporting acetone carboxylation in cell extracts. The sustained carboxylation of acetone in cell extracts required the addition of an ATP-regenerating system consisting of phosphocreatine and creatine kinase, suggesting that the carboxylation of acetone is coupled to ATP hydrolysis. Together, these studies provide the first demonstration of a CO 2 -dependent pathway of acetone metabolism for a strictly aerobic bacterium and provide direct evidence for the involvement of an ATP-dependent carboxylase in bacterial acetone metabolism.A variety of aerobic and anaerobic bacteria are capable of growth by using acetone as a source of carbon and energy. For some aerobic bacteria, the metabolism of acetone has been proposed to proceed via an O 2 -and reductant-dependent hydroxylation reaction producing acetol-(1-hydroxyacetone) as the initial product (4,12,21,23). For anaerobic bacteria, the metabolism of acetone has been proposed to proceed via a CO 2 -dependent carboxylation reaction producing acetoacetate as the initial product as shown in the following equation (2,10,11,13,14,(16)(17)(18): CH 3 COCH 3 ϩ CO 2 3CH 3 COCH 2 COO Ϫ . The carboxylation of acetone is the reverse of acetoacetate decarboxylation, a terminal reaction catalyzed by acetoacetate decarboxylases in certain fermentative bacteria of the genus Clostridium (6, 26).Acetoacetate decarboxylation represents the thermodynamically favorable direction for...

Epoxide carboxylase from Xanthobacter strain Py2 catalyzes the reductant-and NAD ؉ -dependent carboxylation of aliphatic epoxides to ␤-keto acids. Epoxide carboxylase from Xanthobacter strain Py2 has been resolved from cell extracts by anion-exchange chromatography into three protein components, designated I, II, and III, that are obligately required for functional reconstitution of epoxide carboxylase activity. Component II has been purified to homogeneity on the basis of its ability to complement components I and III in restoring epoxide carboxylase activity. Purified component II had a specific activity for epoxide carboxylation of 41.8 mU ⅐ min ؊1 ⅐ mg ؊1 when components I and III were present at saturating levels. The biochemical properties of component II reveal that it is the flavin-containing NADPH:disulfide oxidoreductase that was recently shown by other means to be associated with epoxide degradation activity in Xanthobacter strain Py2 (J. Swaving, J. A. M. de Bont, A. Westphal, and A. Dekok, J. Bacteriol. 178:6644-6646, 1996). The rate of epoxide carboxylation was dependent on the relative concentrations of the three carboxylase components. At fixed concentrations of two of the components, epoxide carboxylation rates were saturated in a hyperbolic fashion by increasing the concentration of the third variable component. Methylepoxypropane has been characterized as a time-dependent, irreversible inactivator of epoxide carboxylase activity that is proposed to be a mechanism-based inactivator of the enzyme. The addition of component I, but not that of component II or III, to methylepoxypropane-inactivated cell extracts restored epoxide carboxylase activity, suggesting that component I contains the epoxide binding and activation sites.There is considerable interest in biological mechanisms for the degradation of aliphatic epoxides due to their toxic, mutagenic, and potential carcinogenic properties (4, 13). A novel strategy for epoxide metabolism has recently been demonstrated for Xanthobacter strain Py2, a gram-negative bacterium which is able to grow at the expense of aliphatic alkenes and epoxides such as propylene and epoxypropane (propylene oxide) (6). The metabolism of epoxypropane was shown to proceed by a CO 2 -dependent carboxylation reaction that resulted in the formation of acetoacetate as the product (10). In the absence of CO 2 , the epoxide-converting enzyme catalyzed the isomerization of terminal and internal epoxides to the corresponding ketones (e.g., epoxypropane to acetone and 2,3-epoxybutane to methylethyl ketone), although these reactions are apparently not of physiological significance (9). The epoxideconverting enzyme is thus both an epoxide isomerase and carboxylase, the nature of the reaction depending on the availability of the cosubstrate CO 2 .Initial studies of Xanthobacter epoxide carboxylase/isomerase suggest a novel and complex catalytic mechanism. In cell extracts, both the isomerase and carboxylase activities are dependent on the addition of NAD ϩ and a reductant (e.g., di...