Alkene monooxygenase from Xanthobacter strain Py2 is an inducible enzyme that catalyzes the O 2 -and NADHdependent epoxidation of short chain (C 2 to C 6 ) alkenes to their corresponding epoxides as the initial step in the utilization of aliphatic alkenes as carbon and energy sources. In the present study, alkene monooxygenase has been resolved from the soluble fraction of cell-free extracts into four components, each of which has been purified to homogeneity, that are obligately required for alkene epoxidation activity. The four required components are 1) a monomeric 35.5-kDa protein containing 1 mol of FAD and a probable 2Fe-2S center; 2) a 13.3-kDa ferredoxin containing a Rieske-type 2Fe-2S cluster; 3) an 11-kDa monomeric protein that contains no detectable cofactors; and 4) a 212-kDa ␣ 2 ␤ 2 ␥ 2 multimeric protein containing four atoms of nonheme iron. The 35.5-kDa protein has been characterized as an NADH reductase. The physiological electron acceptor for the reductase was the Rieske-type ferredoxin, which is proposed to be an intermediate electron carrier between the reductase and terminal catalytic component of the system. The 212-kDa protein was specifically inactivated in cell-free extracts by the mechanism-based inactivator propyne, suggesting that it is the catalytic component and contains the active site(s) for O 2 activation and alkene epoxidation. The subunit structure and metal analysis of this component suggest that it contains two diiron centers, one for each ␣␤␥ protomeric unit. No specific enzymatic activities could be assigned for the 11-kDa protein, but this component was obligately required for steady-state alkene epoxidation. The alkene monooxygenase components were expressed during growth of Xanthobacter Py2 on aliphatic alkenes or epoxides and repressed during growth on other carbon sources. The electron transfer components of alkene monooxygenase were highly specific: other reductase activities present in Xanthobacter were incapable of transferring electrons to the Rieske-type ferredoxin or substituting for the reductase in the alkene monooxygenase complex. Likewise, other bacterial and plant ferredoxins were unable to substitute for the Riesketype ferredoxin in mediating electron transfer to the oxygenase. The biochemical properties of alkene monooxygenase described in this study suggest that this enzyme combines elements of both the well-characterized aromatic dioxygenase (two-component electron transfer scheme) and methane monooxygenase (small regulatory protein and diiron oxygenase) multicomponent enzyme systems.Xanthobacter strain Py2 is one of several bacteria capable of aerobic growth using aliphatic alkenes (e.g. ethylene, propylene, and butylene) as sources of carbon and energy (1, 2). In these alkene-metabolizing bacteria, alkenes are oxidized to epoxides in reactions catalyzed by alkene monooxygenases as illustrated in Equation 1.In Xanthobacter strain Py2, epoxides are further metabolized by a CO 2 -dependent carboxylation reaction, catalyzed by an epoxide carboxylase, that ...

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...

Evidence for a requirement for CO 2 in the productive metabolism of aliphatic alkenes and epoxides by the propylene-oxidizing bacterium Xanthobacter strain Py2 is presented. In the absence of CO 2 , whole-cell suspensions of propylene-grown cells catalyzed the isomerization of propylene oxide (epoxypropane) to acetone. In the presence of CO 2 , no acetone was produced. Acetone was not metabolized by suspensions of propylene-grown cells, in either the absence or presence of CO 2 . The degradation of propylene and propylene oxide by propylene-grown cells supported the fixation of 14 CO 2 into cell material, and the time course of 14 C 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 14 CO 2 fixation. With propylene oxide as the substrate, the concentration dependence of 14 CO 2 fixation exhibited saturation kinetics, and at saturation, 0.9 mol of CO 2 was fixed per mol of propylene oxide consumed. Cultures grown with propylene in a nitrogen-deficient medium supplemented with NaH 13 CO 3 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-␤-hydroxybutyrate. No specific label incorporation was observed when cells were cultured with glucose or n-propanol as a carbon source. The depletion of CO 2 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 CO 2 , is released as acetone.In recent years bacteria which are capable of aerobic growth with aliphatic alkenes and epoxides as carbon and energy sources have been isolated (6). One such bacterium is Xanthobacter strain Py2, which was isolated with propylene as a source of carbon and energy (19). In this bacterium and other alkene-oxidizing bacteria, the metabolism of alkenes is initiated by monooxygenases which catalyze the O 2 -and reductantdependent oxidation of the alkenes to the corresponding epoxides, as illustrated for the substrate propylene and product propylene oxide in the following equation: propylene ϩ O 2 ϩ NADH ϩ H ϩ 3propylene oxide ϩ H 2 O ϩ NAD ϩ . The epoxides thus formed are further metabolized enzymatically, although the mechanisms and pathways involved remain largely uncharacterized. There is considerable interest in biological mechanisms for the disposal of aliphatic epoxides because of the toxicity, mutagenicity, and potential carcinogenicity of these compounds (3,20). Epoxides are highly electrophilic molecules, and their detrimental properties arise from their ability to covalently modify cellular macromolecules, including proteins and DNA (3,20). Epoxides are formed through the cytochrome P450-catalyzed epoxidation of alkenes (11) and are also synthesized in large quantities by the pet...

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 beta-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.

Proton NMR spectra of the Rieske-type ferredoxin from Xanthobacter strain Py2 were recorded in both H2O and D2O buffered solutions at pH 7.2. Several well-resolved hyperfine-shifted 1H NMR signals were observed in the 90 to -20 ppm chemical shift range. Comparison of spectra recorded in H2O and D2O buffered solutions indicated that the signals at -11.4 (L) and -15.5 (M) ppm were solvent-exchangeable and thus were assigned to the two histidine N epsilon 2H protons. The remaining observed signals were assigned based upon chemical shift, T1 values, and one-dimensional nuclear Overhauser effect (nOe) saturation transfer experiments to either C beta H or C alpha H protons of cluster cysteinyl or histidine ligands. Upon oxidation of the [2Fe-2S] cluster, only two broad resonances were observed, indicating that the two Fe(III) ions are strongly antiferromagnetically coupled. In addition, the temperature dependence of each observed hyperfine-shifted signal in the reduced state was determined, providing information about the magnetic properties of the [2Fe-2S]1- cluster. Fits of the temperature data observed for each resonance to equations describing the hyperfine shift with their Boltzmann weighting factors provided a delta EL value of 185 +/- 26 cm-1 which, in turn, gives -2J as 124 cm-1. These data indicate that the two iron centers in the reduced [2Fe-2S] Rieske-type center are moderately antiferromagnetically coupled. The combination of these data with the available spectroscopic and crystallographic results for Rieske-type proteins has provided new insights into the role of the Rieske-type protein from Xanthobacter strain Py2 in alkene oxidation.