Involvement of an ATP-dependent carboxylase in a CO2-dependent pathway of acetone metabolism by Xanthobacter strain Py2.

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 CO2-dependent pathway of acetone metabolism for this bacterium. The depletion of CO2 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 CO2 and was prevented by the depletion of CO2. The degradation of acetone by acetone-grown cell suspensions supported the fixation of 14CO2 into acid-stable products, while the degradation of glucose or beta-hydroxybutyrate did not. Cultures grown with acetone in a nitrogen-deficient medium supplemented with NaH13CO3 specifically incorporated 13C-label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-beta-hydroxybutyrate. Cell extracts prepared from acetone-grown cells catalyzed the CO2- 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 CO2-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.     

Carbon dioxide fixation in the metabolism of propylene and propylene oxide by Xanthobacter strain Py2.

Evidence for a requirement for CO2 in the productive metabolism of aliphatic alkenes and epoxides by the propylene-oxidizing bacterium Xanthobacter strain Py2 is presented. In the absence of CO2, whole-cell suspensions of propylene-grown cells catalyzed the isomerization of propylene oxide (epoxypropane) to acetone. In the presence of CO2, no acetone was produced. Acetone was not metabolized by suspensions of propylene-grown cells, in either the absence or presence of CO2. The degradation of propylene and propylene oxide by propylene-grown cells supported the fixation of 14CO2 into cell material, and the time course of 14C fixation correlated with the time course of propylene and propylene oxide degradation. The degradation of glucose and propionaldehyde by propylene-grown or glucose-grown cells did not support significant 14CO2 fixation. With propylene oxide as the substrate, the concentration dependence of 14CO2 fixation exhibited saturation kinetics, and at saturation, 0.9 mol of CO2 was fixed per mol of propylene oxide consumed. Cultures grown with propylene in a nitrogen-deficient medium supplemented with NaH13CO3 specifically incorporated 13C label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-beta-hydroxybutyrate. No specific label incorporation was observed when cells were cultured with glucose or n-propanol as a carbon source. The depletion of CO2 from cultures grown with propylene, but not glucose or n-propanol, inhibited bacterial growth. We propose that propylene oxide metabolism in Xanthobacter strain Py2 proceeds by terminal carboxylation of an isomerization intermediate, which, in the absence of CO2, is released as acetone.     

Aliphatic and chlorinated alkenes and epoxides as inducers of alkene monooxygenase and epoxidase activities in Xanthobacter strain Py2.

The inducible nature of the alkene oxidation system of Xanthobacter strain Py2 has been investigated. Cultures grown with glucose as the carbon source did not contain detectable levels of alkene monooxygenase or epoxidase, two key enzymes of alkene and epoxide metabolism. Upon addition of propylene to glucose-grown cultures, alkene monooxygenase and epoxidase activities increased and after an 11-h induction period reached levels of specific activity comparable to those in propylene-grown cells. Addition of chloramphenicol or rifampin prevented the increase in the enzyme activities. Comparison of the banding patterns of proteins present in cell extracts revealed that polypeptides with molecular masses of 43, 53, and 57 kDa accumulate in propylene-grown but not glucose-grown cells. Pulse-labeling of glucose-grown cells with [35S]methionine and [35S]cysteine revealed that the 43-, 53-, and 57-kDa proteins, as well as two additional polypeptides with molecular masses of 12 and 21 kDa, were newly synthesized upon exposure of cells to propylene or propylene oxide. The addition to glucose-grown cells of a variety of other aliphatic and chlorinated alkenes and epoxides, including ethylene, vinyl chloride (1-chloroethylene), cis- and trans-1,2-dichloroethylene, 1-chloropropylene, 1,3-dichloropropylene, 1-butylene, trans-2-butylene, isobutylene, ethylene oxide, epichlorohydrin (3-chloro-1,2-epoxypropane), 1,2-epoxybutane, cis- and trans-2,3-epoxybutane, and isobutylene oxide stimulated the synthesis of the five propylene-inducible polypeptides as well as increases in alkene monooxygenase and epoxidase activities. In contrast, acetylene, and a range of aliphatic and chlorinated alkanes, did not stimulate the synthesis of the propylene-inducible polypeptides or the increase in alkene monooxygenase and epoxidase activities.     

Evidence that a linear megaplasmid encodes enzymes of aliphatic alkene and epoxide metabolism and coenzyme M (2-mercaptoethanesulfonate) biosynthesis in Xanthobacter strain Py2

The bacterial metabolism of propylene proceeds by epoxidation to epoxypropane followed by a sequence of three reactions resulting in epoxide ring opening and carboxylation to form acetoacetate. Coenzyme M (2-mercaptoethanesulfonic acid) (CoM) plays a central role in epoxide carboxylation by serving as the nucleophile for epoxide ring opening and the carrier of the C(3) unit that is ultimately carboxylated to acetoacetate, releasing CoM. In the present work, a 320-kb linear megaplasmid has been identified in the gram-negative bacterium Xanthobacter strain Py2, which contains the genes encoding the key enzymes of propylene oxidation and epoxide carboxylation. Repeated subculturing of Xanthobacter strain Py2 under nonselective conditions, i.e., with glucose or acetate as the carbon source in the absence of propylene, resulted in the loss of the propylene-positive phenotype. The propylene-negative phenotype correlated with the loss of the 320-kb linear megaplasmid, loss of induction and expression of alkene monooxgenase and epoxide carboxylation enzyme activities, and the loss of CoM biosynthetic capability. Sequence analysis of a hypothetical protein (XecG), encoded by a gene located downstream of the genes for the four enzymes of epoxide carboxylation, revealed a high degree of sequence identity with proteins of as-yet unassigned functions in the methanogenic archaea Methanobacterium thermoautotrophicum and Methanococcus jannaschii and in Bacillus subtilis. The M. jannaschii homolog of XecG, MJ0255, is located next to a gene, MJ0256, that has been shown to encode a key enzyme of CoM biosynthesis (M. Graupner, H. Xu, and R. H. White, J. Bacteriol. 182: 4862-4867, 2000). We propose that the propylene-positive phenotype of Xanthobacter strain Py2 is dependent on the selective maintenance of a linear megaplasmid containing the genes for the key enzymes of alkene oxidation, epoxide carboxylation, and CoM biosynthesis.     

Identification and Characterization of Epoxide Carboxylase Activity in Cell Extracts of Nocardia corallina B276

The metabolism of aliphatic epoxides (epoxyalkanes) by the alkene-utilizing actinomycete Nocardia corallina B276 was investigated. Suspensions of N. corallina cells grown with propylene as the carbon source readily degraded propylene and epoxypropane, while suspensions of glucose-grown cells did not. The addition of propylene and epoxypropane to glucose-grown cells resulted in a time-dependent increase in propylene- and epoxypropane-degrading activities that was prevented by the addition of rifampin and chloramphenicol. The expression of alkene- and epoxide-degrading activities was correlated with the high-level expression of several polypeptides not present in extracts of glucose-grown cells. Epoxypropane and epoxybutane degradation by propylene-grown cell suspensions of N. corallina was stimulated by the addition of CO₂ and inhibited by the depletion of CO₂. Cell extracts catalyzed the carboxylation of epoxypropane to form acetoacetate in a reaction that was dependent on the addition of CO₂, NAD⁺, and a reductant (NADPH or dithiothreitol). In the absence of CO₂, epoxypropane was isomerized by cell extracts to form acetone at a rate approximately 10-fold lower than the rate of epoxypropane carboxylation. Methylepoxypropane was found to be a time-dependent, irreversible inactivator of epoxyalkane-degrading activity. These properties demonstrate that epoxyalkane metabolism in N. corallina occurs by a carboxylation reaction forming β-keto acids as products and provide evidence for the involvement in this reaction of an epoxide carboxylase with properties and cofactor requirements similar to those of the four-component epoxide carboxylase enzyme system of the gram-negative bacterium Xanthobacter strain Py2 (J. R. Allen and S. A. Ensign, J. Biol. Chem. 272:32121–32128, 1997). The addition of epoxide carboxylase component I from Xanthobacter strain Py2 to methylepoxypropane-inactivated N. corallina extracts restored epoxide carboxylase activity, and the addition of epoxide carboxylase component II from Xanthobacter Py2 to active N. corallina extracts stimulated epoxide isomerase rates to the same levels observed with the purified Xanthobacter system. Antibodies raised against Xanthobacter strain Py2 epoxide carboxylase component I cross-reacted with a polypeptide in propylene-grown N. corallina extracts with the same molecular weight as component I but did not cross-react with glucose-grown extracts. Together, these results suggest a common pathway of epoxyalkane metabolism for phylogenetically distinct bacteria that involves CO₂ fixation and the activity of a multicomponent epoxide carboxylase enzyme system.     

Characterization of a New Pathway for Epichlorohydrin Degradation by Whole Cells of Xanthobacter Strain Py2

The degradation of epichlorohydrin (3-chloropropylene oxide or 1-chloro-2,3-epoxypropane) by whole-cell suspensions of Xanthobacter strain Py2 was investigated. Cell suspensions prepared from cultures grown with propylene as the carbon source readily degraded epichlorohydrin. The ability to degrade epichlorohydrin correlated with the expression of enzymes involved in alkene and epoxide metabolism, since cell suspensions prepared from cultures grown with glucose or acetone, in which the enzymes of alkene and epoxide oxidation are not expressed, did not degrade epichlorohydrin. The alkene monooxygenase-specific inhibitor propyne had no effect on the degradation of epichlorohydrin, demonstrating that alkene monooxygenase is not involved in epichlorohydrin conversion. The interaction of epichlorohydrin and epibromohydrin with the epoxidase which catalyzes aliphatic epoxide conversions was established by showing that the epihalohydrins were specific and potent inhibitors of propylene oxide-dependent O(inf2) consumption by cell suspensions. The rates of degradation of epoxides in whole-cell suspensions decreased in the series propylene oxide > epifluorohydrin > epichlorohydrin > epibromohydrin. The pathway of epichlorohydrin degradation was investigated and found to proceed with stoichiometric dechlorination of epichlorohydrin. The first detectable product of epichlorohydrin degradation was chloroacetone. Chloroacetone was further degraded by the cell suspensions, and in the process, acetone was formed as a nonstoichiometric product. Acetone was further degraded by the cell suspensions with enzymes apparently induced by the accumulation of acetone. The metabolism of allyl chloride (3-chloropropylene) by propylene-grown cells was initiated by alkene monooxygenase and proceeded through epichlorohydrin, chloroacetone, and acetone as intermediate degradation products. These studies reveal a new pathway for halogenated epoxide degradation which involves halogenated and aliphatic ketones as well as other unidentified intermediates and which is unique from previously characterized hydrolytic degradative pathways.     

Characterization of three protein components required for functional reconstitution of the epoxide carboxylase multienzyme complex from Xanthobacter strain Py2.

Epoxide carboxylase from Xanthobacter strain Py2 catalyzes the reductant- and NAD+-dependent carboxylation of aliphatic epoxides to beta-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 x min(-1) x 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.     

Structural basis for CO2 fixation by a novel member of the disulfide oxidoreductase family of enzymes, 2-ketopropyl-coenzyme M oxidoreductase/carboxylase

The NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is the terminal enzyme in a metabolic pathway that results in the conversion of propylene to the central metabolite acetoacetate in Xanthobacter autotrophicus Py2. This enzyme is an FAD-containing enzyme that is a member of the NADPH:disulfide oxidoreductase (DSOR) family of enzymes that include glutathione reductase, dihydrolipoamide dehydrogenase, trypanothione reductase, thioredoxin reductase, and mercuric reductase. In contrast to the prototypical reactions catalyzed by members of the DSOR family, the NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase catalyzes the reductive cleavage of the thioether linkage of 2-ketopropyl-coenzyme M, and the subsequent carboxylation of the ketopropyl cleavage product, yielding the products acetoacetate and free coenzyme M. The structure of 2-KPCC reveals a unique active site in comparison to those of other members of the DSOR family of enzymes and demonstrates how the enzyme architecture has been adapted for the more sophisticated biochemical reaction. In addition, comparison of the structures in the native state and in the presence of bound substrate indicates the binding of the substrate 2-ketopropyl-coenzyme M induces a conformational change resulting in the collapse of the substrate access channel. The encapsulation of the substrate in this manner is reminiscent of the conformational changes observed in the well-characterized CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxidase (Rubisco).     

Characterization of 2-bromoethanesulfonate as a selective inhibitor of the coenzyme m-dependent pathway and enzymes of bacterial aliphatic epoxide metabolism

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.     

Characterization of five catalytic activities associated with the NADPH:2-ketopropyl-coenzyme M [2-(2-ketopropylthio)ethanesulfonate] oxidoreductase/carboxylase of the Xanthobacter strain Py2 epoxide carboxylase system

The bacterial metabolism of propylene proceeds by epoxidation to epoxypropane followed by carboxylation to acetoacetate. Epoxypropane carboxylation is a minimetabolic pathway that requires four enzymes, NADPH, NAD(+), and coenzyme M (CoM; 2-mercaptoethanesulfonate) and occurs with the overall reaction stoichiometry: epoxypropane + CO(2) + NADPH + NAD(+) + CoM --> acetoacetate + H(+) + NADP(+) + NADH + CoM. The terminal enzyme of the pathway is NADPH:2-ketopropyl-CoM [2-(2-ketopropylthio)ethanesulfonate] oxidoreductase/carboxylase (2-KPCC), an FAD-containing enzyme that is a member of the NADPH:disulfide oxidoreductase family of enzymes and that catalyzes the reductive cleavage and carboxylation of 2-ketopropyl-CoM to form acetoacetate and CoM according to the reaction: 2-ketopropyl-CoM + NADPH + CO(2) --> acetoacetate + NADP(+) + CoM. In the present work, 2-KPCC has been characterized with respect to the above reaction and four newly discovered partial reactions of relevance to the catalytic mechanism, and each of which requires the formation of a stabilized enolacetone intermediate. These four reactions are (1) NADPH-dependent cleavage and protonation of 2-ketopropyl-CoM to form NADP(+), CoM, and acetone, a reaction analogous to the physiological reaction but in which H(+) is the electrophile; (2) NADP(+)-dependent synthesis of 2-ketopropyl-CoM from CoM and acetoacetate, the reverse of the physiologically important forward reaction; (3) acetoacetate decarboxylation to form acetone and CO(2); and (4) acetoacetate/(14)CO(2) exchange to form (14)C(1)-acetoacetate and CO(2). Acetoacetate decarboxylation and (14)CO(2) exchange occurred independent of NADP(H) and CoM, demonstrating that these substrates are not central to the mechanism of enolate generation and stabilization. 2-KPCC did not uncouple NADPH oxidation or NADP(+) reduction from the reactions involving cleavage or formation of 2-ketopropyl-CoM. N-Ethylmaleimide inactivated the reactions forming/using 2-ketopropyl-CoM but did not inactivate acetoacetate decarboxylation or (14)CO(2) exchange reactions. The biochemical characterization of 2-KPCC and the associated five catalytic activities has allowed the formulation of an unprecedented mechanism of substrate activation and carboxylation that involves NADPH oxidation, a redox active disulfide, thiol-mediated reductive cleavage of a C-S thioether bond, the formation of a CoM:cysteine mixed disulfide, and enolacetone stabilization.     

Getting a Handle on the Role of Coenzyme M in Alkene Metabolism

Summary: Coenzyme M (2-mercaptoethanesulfonate; CoM) is one of several atypical cofactors discovered in methanogenic archaea which participate in the biological reduction of CO₂ to methane. Elegantly simple, CoM, so named for its role as a methyl carrier in all methanogenic archaea, is the smallest known organic cofactor. It was thought that this cofactor was used exclusively in methanogenesis until it was recently discovered that CoM is a key cofactor in the pathway of propylene metabolism in the gram-negative soil microorganism Xanthobacter autotrophicus Py2. A four-step pathway requiring CoM converts propylene and CO₂ to acetoacetate, which feeds into central metabolism. In this process, CoM is used to activate and convert highly electrophilic epoxypropane, formed from propylene epoxidation, into a nucleophilic species that undergoes carboxylation. The unique properties of CoM provide a chemical handle for orienting compounds for site-specific redox chemistry and stereospecific catalysis. The three-dimensional structures of several of the enzymes in the pathway of propylene metabolism in defined states have been determined, providing significant insights into both the enzyme mechanisms and the role of CoM in this pathway. These studies provide the structural basis for understanding the efficacy of CoM as a handle to direct organic substrate transformations at the active sites of enzymes.     

Roles of the Redox-Active Disulfide and Histidine Residues Forming a Catalytic Dyad in Reactions Catalyzed by 2-Ketopropyl Coenzyme M Oxidoreductase/Carboxylase

NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC), an atypical member of the disulfide oxidoreductase (DSOR) family of enzymes, catalyzes the reductive cleavage and carboxylation of 2-ketopropyl-coenzyme M [2-(2-ketopropylthio)ethanesulfonate; 2-KPC] to form acetoacetate and coenzyme M (CoM) in the bacterial pathway of propylene metabolism. Structural studies of 2-KPCC from Xanthobacter autotrophicus strain Py2 have revealed a distinctive active-site architecture that includes a putative catalytic triad consisting of two histidine residues that are hydrogen bonded to an ordered water molecule proposed to stabilize enolacetone formed from dithiol-mediated 2-KPC thioether bond cleavage. Site-directed mutants of 2-KPCC were constructed to test the tenets of the mechanism proposed from studies of the native enzyme. Mutagenesis of the interchange thiol of 2-KPCC (C82A) abolished all redox-dependent reactions of 2-KPCC (2-KPC carboxylation or protonation). The air-oxidized C82A mutant, as well as wild-type 2-KPCC, exhibited the characteristic charge transfer absorbance seen in site-directed variants of other DSOR enzymes but with a pK_a value for C87 (8.8) four units higher (i.e., four orders of magnitude less acidic) than that for the flavin thiol of canonical DSOR enzymes. The same higher pK_a value was observed in native 2-KPCC when the interchange thiol was alkylated by the CoM analog 2-bromoethanesulfonate. Mutagenesis of the flavin thiol (C87A) also resulted in an inactive enzyme for steady-state redox-dependent reactions, but this variant catalyzed a single-turnover reaction producing a 0.8:1 ratio of product to enzyme. Mutagenesis of the histidine proximal to the ordered water (H137A) led to nearly complete loss of redox-dependent 2-KPCC reactions, while mutagenesis of the distal histidine (H84A) reduced these activities by 58 to 76%. A redox-independent reaction of 2-KPCC (acetoacetate decarboxylation) was not decreased for any of the aforementioned site-directed mutants. We interpreted and rationalized these results in terms of a mechanism of catalysis for 2-KPCC employing a unique hydrophobic active-site architecture promoting thioether bond cleavage and enolacetone formation not seen for other DSOR enzymes.     

Heterologous expression of bacterial Epoxyalkane:Coenzyme M transferase and inducible coenzyme M biosynthesis in Xanthobacter strain Py2 and Rhodococcus rhodochrous B276

Coenzyme M (CoM) (2-mercaptoethanesulfonic acid) biosynthesis is shown to be coordinately regulated with the expression of the enzymes of alkene and epoxide metabolism in the propylene-oxidizing bacteria Xanthobacter strain Py2 and Rhodococcus rhodochrous strain B276. These results provide the first evidence for the involvement of CoM in propylene metabolism by R. rhodochrous and demonstrate for the first time the inducible nature of eubacterial CoM biosynthesis.     

Epoxidation of alkenes by alkene-grown Xanthobacter spp.

Summary Newly isolated Xanthobacter spp. were able to grow on the gaseous alkenes like ethene, propene, 1-butene and 1,3-butadiene. Resting-cell suspensions of propene-, 1-butene- or 1,3-butadiene-grown Xanthobacter Py10 accumulated 1,2-epoxyethane from ethene. Ethene-grown Xanthobacter Py10 did not produce any 1,2-epoxyalkane from the alkenes tested. Furthermore, propenegrown Xanthobacter Py2 accumulated 2,3-epoxybutane from trans-butene and cis-butene but did not form epoxides from other substrates tested.     

New roles for CO2 in the microbial metabolism of aliphatic epoxides and ketones

Short-chain aliphatic epoxides and ketones are two classes of toxic organic compounds formed biogenically and anthropogenically. In spite of their toxicity, these compounds are utilized as primary carbon and energy sources or are generated as intermediate metabolites in the metabolism of other compounds (e.g., alkenes, alkanes, and secondary alcohols) by a number of diverse bacteria. One bacterium capable of using both classes of compounds is the gram-negative aerobe Xanthobacter strain Py2. Studies of epoxide and ketone (acetone) metabolism by Xanthobacter strain Py2 have revealed a central role for CO₂ in these processes. Both classes of compounds are metabolized by carboxylation reactions that produce β-keto acids as products. The epoxide- and ketone-converting enzymes are distinct carboxylases with molecular properties and cofactor requirements unprecedented for other carboxylases. Epoxide carboxylase is a four-component multienzyme complex that requires NADPH and NAD⁺ as cofactors. In the course of epoxide carboxylation, a transhydrogenation reaction occurs wherein NADPH undergoes oxidation and NAD⁺ undergoes reduction. Acetone carboxylase is a multimeric (three-subunit) ATP-dependent enzyme that forms AMP and inorganic phosphate as ATP hydrolysis products in the course of acetone carboxylation. Recent studies have demonstrated that acetone metabolism in diverse anaerobic bacteria (sulfate reducers, denitrifiers, phototrophs, and fermenters) also proceeds by carboxylation reactions. ATP-dependent acetone carboxylase activity has been demonstrated in cell-free extracts of the anaerobic acetone-utilizers Rhodobacter capsulatus, Rhodomicrobium vannielii, and Thiosphaera pantotropha. These studies have identified new roles for CO₂ as a cosubstrate in the metabolism of two classes of important xenobiotic compounds. In addition, two new classes of carboxylases have been identified, the investigation of which promises to reveal new insights into biological strategies for the fixation of CO₂ to organic substrates. Received: 13 August 1997 / Accepted: 6 October 1997     

A novel type of pyridine nucleotide-disulfide oxidoreductase is essential for NAD+- and NADPH-dependent degradation of epoxyalkanes by Xanthobacter strain Py2.

Epoxide degradation in cell extracts of Xanthobacter strain Py2 has been reported to be dependent on NAD+ and dithiols. This multicomponent system has now been fractionated. A key protein encoded by a DNA fragment complementing a Xanthobacter strain Py2 mutant unable to degrade epoxides was purified and analyzed. This NADP-dependent protein, a novel type of pyridine nucleotide-disulfide oxidoreductase, is essential for epoxide degradation. NADPH, acting as the physiological cofactor, replaced the dithiols in epoxide conversion.     

Oxidation of propene and 1-butene byMethylococcus capsulatus andMethylosinus trichosporium

Summary Methane-grown cells ofMethylococcus capsulatus andMethylosinus trichosporium readily oxidized propene and various isomers of butene to their respective epoxides. When examined in a proton NMR spectrum using tris([3-trifluoromethylhydroxymethylene]-d-camphorato), europium III derivative as an optically active chemical shift reagent, the products propylene oxide and 1,2-epoxybutane were found to contain equal amounts of both isomers. Methane-grown cells of both bacteria had considerable levels of reducing equivalents to catalyze the epoxidation of gaseous olefins. Cells depleted of reductants catalyzed the oxidation in the presence of low levels of methanol or formaldehyde with a stoichiometry of about 2:1. The rates of epoxidation of propene and 1-butene in a continuous reactor were 2–3-times that of a batch-wise reaction; the epoxidation activity, however, was lost within 3 h. The inactivation was attributed to the reactivity of the accumulated epoxides in the reactor. Propene and 1-butene oxidation by both bacteria were drastically inhibited by the respective products. Thus, the major problem in the application of microorganisms for production of epoxides from gaseous olefins is the rapid separation of the reactive products.     

Cometabolic degradation of chlorinated alkenes by alkene monooxygenase in a propylene-grown Xanthobacter strain.

Propylene-grown Xanthobacter cells (strain Py2) degraded several chlorinated alkenes of environmental concern, including trichloroethylene, 1-chloroethylene (vinyl chloride), cis- and trans-1,2-dichloroethylene, 1,3-dichloropropylene, and 2,3-dichloropropylene. 1,1-Dichloroethylene was not degraded efficiently, while tetrachloroethylene was not degraded. The role of alkene monooxygenase in catalyzing chlorinated alkene degradations was established by demonstrating that glucose-grown cells which lack alkene monooxygenase and propylene-grown cells in which alkene monooxygenase was selectively inactivated by propyne were unable to degrade the compounds. C2 and C3 chlorinated alkanes were not oxidized by alkene monooxygenase, but a number of these compounds were inhibitors of propylene and ethylene oxidation, suggesting that they compete for binding to the enzyme. A number of metabolites enhanced the rate of degradation of chlorinated alkenes, including propylene oxide, propionaldehyde, and glucose. Propylene stimulated chlorinated alkene oxidation slightly when present at a low concentration but became inhibitory at higher concentrations. Toxic effects associated with chlorinated alkene oxidations were determined by measuring the propylene oxidation and propylene oxide-dependent O2 uptake rates of cells previously incubated with chlorinated alkenes. Compounds which were substrates for alkene monooxygenase exhibited various levels of toxicity, with 1,1-dichloroethylene and trichloroethylene being the most potent inactivators of propylene oxidation and 1,3- and 2,3-dichloropropylene being the most potent inactivators of propylene oxide-dependent O2 uptake. No toxic effects were seen when cells were incubated with chlorinated alkenes anaerobically, indicating that the product(s) of chlorinated alkene oxidation mediates toxicity.     

Cometabolic degradation of chlorinated alkenes by alkene monooxygenase in a propylene-grown Xanthobacter strain.

Propylene-grown Xanthobacter cells (strain Py2) degraded several chlorinated alkenes of environmental concern, including trichloroethylene, 1-chloroethylene (vinyl chloride), cis- and trans-1,2-dichloroethylene, 1,3-dichloropropylene, and 2,3-dichloropropylene. 1,1-Dichloroethylene was not degraded efficiently, while tetrachloroethylene was not degraded. The role of alkene monooxygenase in catalyzing chlorinated alkene degradations was established by demonstrating that glucose-grown cells which lack alkene monooxygenase and propylene-grown cells in which alkene monooxygenase was selectively inactivated by propyne were unable to degrade the compounds. C2 and C3 chlorinated alkanes were not oxidized by alkene monooxygenase, but a number of these compounds were inhibitors of propylene and ethylene oxidation, suggesting that they compete for binding to the enzyme. A number of metabolites enhanced the rate of degradation of chlorinated alkenes, including propylene oxide, propionaldehyde, and glucose. Propylene stimulated chlorinated alkene oxidation slightly when present at a low concentration but became inhibitory at higher concentrations. Toxic effects associated with chlorinated alkene oxidations were determined by measuring the propylene oxidation and propylene oxide-dependent O2 uptake rates of cells previously incubated with chlorinated alkenes. Compounds which were substrates for alkene monooxygenase exhibited various levels of toxicity, with 1,1-dichloroethylene and trichloroethylene being the most potent inactivators of propylene oxidation and 1,3- and 2,3-dichloropropylene being the most potent inactivators of propylene oxide-dependent O2 uptake. No toxic effects were seen when cells were incubated with chlorinated alkenes anaerobically, indicating that the product(s) of chlorinated alkene oxidation mediates toxicity.     

The human gastric pathogen <Emphasis Type="Italic">Helicobacter pylori</Emphasis> has a potential acetone carboxylase that enhances its ability to colonize mice

Background  

Helicobacter pyloricolonizes the human stomach and is the etiological agent of peptic ulcer disease. All threeH. pyloristrains that have been sequenced to date contain a potential operon whose products share homology with the subunits of acetone carboxylase (encoded byacxABC) fromXanthobacter autotrophicusstrain Py2 andRhodobacter capsulatusstrain B10. Acetone carboxylase catalyzes the conversion of acetone to acetoacetate. Genes upstream of the putativeacxABCoperon encode enzymes that convert acetoacetate to acetoacetyl-CoA, which is metabolized further to generate two molecules of acetyl-CoA.