The Alkene Monooxygenase from Xanthobacter Strain Py2 Is Closely Related to Aromatic Monooxygenases and Catalyzes Aromatic Monohydroxylation of Benzene, Toluene, and Phenol

Abstract

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 α-subunit (XamoA); aamB, encoding the 88-residue oxygenase γ-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 β-subunit (XamoE); andaamF, encoding the 327-residue reductase (XamoF). A sequence with >60% concurrence with the consensus sequence of ς54 (RpoN)-dependent promoters was identified upstream of the aamA gene. Detailed comparison of XamoA with the oxygenase α-subunits from aromatic monooxygenases, phenol hydroxylases, methane monooxygenase, and the alkene monooxygenase fromRhodococcus 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, Xanthobacterstrain 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 ofo-, 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 studiesXanthobacter 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 theaam 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.

Identification and characterisation of isoprene‐degrading bacteria in an estuarine environment

“…All positions containing gaps and missing data were eliminated, and there were a total of 535 positions in the final dataset. B. Phylogenetic relationship of known isoprene-degrading strains based on isoA sequences, together with alkene monooxygenase (xamoA) from Xanthobacter autotrophicus Py2 (Zhou et al, 1999). The tree was drawn in MEGA6 using the Maximum Likelihood method based on an alignment of isoA sequences.…”

Evolution of the soluble diiron monooxygenases

“…Both enzymes are of commercial interest because of their ability to produce optically active epoxides from some substrates [105,106]. The speci¢city of the Aam alkene monooxygenase overlaps with that of the aromatic monooxygenases in the group, having been shown not only to oxidize unsubstituted and chlorinated alkenes up to six carbons, but also benzene, toluene, and phenol [104]. Table 1 lists the soluble diiron monooxygenases that were analyzed for the present study, together with the source organisms, the organization of gene operons that encode the oxygenase subunits for each enzyme, and their genomic location.…”

The oxygenase component of phenol hydroxylase from Acinetobacter radioresistens S13

“…S 13 phenol hydroxylase a component; MMO B, M. capsulatus methane monooxygenase [17]; MMO M, M. trichosporium methane monooxygenase [18]; AMO a, N. corallina alkene monoxygenase; AMO Py2, X.Py2 alkene monoxygenase[36]; Xyl/TMO, P. stutzeri xylene/toluene monooxygenase[6]; T4MO, P. mendocina toluene 4 monooxygenase[37]; T3MO, of one di-iron centre per each (abc) monomer. Iron content determined by the Lovenberg colorimetric method…”