Publications

• The alkene monooxygenase from Xanthobacter strain Py2 is closely related to aromatic monooxygenases and catalyzes aromatic monohydroxylation of benzene, toluene, and phenol.

  Zhou N.Y., Jenkins A., Chan Kwo Chion C.K., Leak D.J.

  The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in cosmid pNY2. Sequencing and analysis of the predicted amino acid sequences indicate that the components o ... >> More

  The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in cosmid pNY2. Sequencing and analysis of the predicted amino acid sequences indicate that the components of Xamo are homologous to those of the aromatic monooxygenases, toluene 2-, 3-, and 4-monooxygenase and benzene monooxygenase, and that the gene order is identical. The genes and predicted polypeptides are aamA, encoding the 497-residue oxygenase alpha-subunit (XamoA); aamB, encoding the 88-residue oxygenase gamma-subunit (XamoB); aamC, encoding the 122-residue ferredoxin (XamoC); aamD, encoding the 101-residue coupling or effector protein (XamoD); aamE, encoding the 341-residue oxygenase beta-subunit (XamoE); and aamF, encoding the 327-residue reductase (XamoF). A sequence with >60% concurrence with the consensus sequence of sigma54 (RpoN)-dependent promoters was identified upstream of the aamA gene. Detailed comparison of XamoA with the oxygenase alpha-subunits from aromatic monooxygenases, phenol hydroxylases, methane monooxygenase, and the alkene monooxygenase from Rhodococcus rhodochrous B276 showed that, despite the overall similarity to the aromatic monooxygenases, XamoA has some distinctive characteristics of the oxygenases which oxidize aliphatic, and particularly alkene, substrates. On the basis of the similarity between Xamo and the aromatic monooxygenases, Xanthobacter strain Py2 was tested and shown to oxidize benzene, toluene, and phenol, while the alkene monooxygenase-negative mutants NZ1 and NZ2 did not. Benzene was oxidized to phenol, which accumulated transiently before being further oxidized. Toluene was oxidized to a mixture of o-, m-, and p-cresols (39.8, 18, and 41.7%, respectively) and a small amount (0.5%) of benzyl alcohol, none of which were further oxidized. In growth studies Xanthobacter strain Py2 was found to grow on phenol and catechol but not on benzene or toluene; growth on phenol required a functional alkene monooxygenase. However, there is no evidence of genes encoding steps in the metabolism of catechol in the vicinity of the aam gene cluster. This suggests that the inducer specificity of the alkene monooxygenase may have evolved to benefit from the naturally broad substrate specificity of this class of monooxygenase and the ability of the host strain to grow on catechol. << Less

  Appl. Environ. Microbiol. 65:1589-1595(1999) [PubMed] [EuropePMC]

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

  Ensign S.A., Hyman M.R., Arp D.J.

  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 degrade ... >> More

  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. << Less

  Appl. Environ. Microbiol. 58:3038-3046(1992) [PubMed] [EuropePMC]

• Alkene monooxygenase from Xanthobacter strain Py2. Purification and characterization of a four-component system central to the bacterial metabolism of aliphatic alkenes.

  Small F.J., Ensign S.A.

  Alkene monooxygenase from Xanthobacter strain Py2 is an inducible enzyme that catalyzes the O2- and NADH-dependent epoxidation of short chain (C2 to C6) alkenes to their corresponding epoxides as the initial step in the utilization of aliphatic alkenes as carbon and energy sources. In the present ... >> More

  Alkene monooxygenase from Xanthobacter strain Py2 is an inducible enzyme that catalyzes the O2- and NADH-dependent epoxidation of short chain (C2 to C6) 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 alpha2beta2gamma2 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 O2 activation and alkene epoxidation. The subunit structure and metal analysis of this component suggest that it contains two diiron centers, one for each alphabetagamma 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 Rieske-type 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. << Less

  J. Biol. Chem. 272:24913-24920(1997) [PubMed] [EuropePMC]