Baeyer-Villiger monooxygenases represent useful biocatalytic tools, as they can catalyze reactions which are difficult to achieve using chemical means. However, only a limited number of these atypical monooxygenases are available in recombinant form. Using a recently described protein sequence motif, a putative Baeyer-Villiger monooxygenase (BVMO) was identified in the genome of the thermophilic actinomycete Thermobifida fusca. Heterologous expression of the respective protein in Escherichia coli and subsequent enzyme characterization showed that it indeed represents a BVMO. The NADPH-dependent and FAD-containing monooxygenase is active with a wide range of aromatic ketones, while aliphatic substrates are also converted. The best substrate discovered so far is phenylacetone (k(cat) = 1.9 s(-1), K(M) = 59 microM). The enzyme exhibits moderate enantioselectivity with alpha-methylphenylacetone (enantiomeric ratio of 7). In addition to Baeyer-Villiger reactions, the enzyme is able to perform sulfur oxidations. Different from all known BVMOs, this newly identified biocatalyst is relatively thermostable, displaying an activity half-life of 1 day at 52 degrees C. This study demonstrates that, using effective annotation tools, genomes can efficiently be exploited as a source of novel BVMOs.

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity. The k cat and K m values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s ؊1 and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK a of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.Halogenated aliphatics constitute an important class of environmental pollutants. Various microorganisms have evolved that are able to degrade some of these compounds and use them as sole sources of carbon and energy. Such organisms are of importance for bioremediation of polluted soil, groundwater, and wastewater. In most cases, specialized enzymes, designated dehalogenases, catalyze the cleavage of the carbonhalogen bonds, which is a key detoxification reaction. Hydrolytic dehalogenases have been studied extensively, which has resulted in detailed insight into the structure and mechanism of several enzymes of this class (8,33). For other dehalogenases, structural and mechanistic data are hardly available.Halohydrin dehalogenases, also referred to as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, occur in the degradation pathways of halopropanols and 1,2-dibromoethane, where they catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, yielding an epoxide, a proton, and a halide ion (7,22,30,31). These enzymes also efficiently catalyze the reverse reaction, the halogenation of epoxides, and the dehalogenation of vicinal chlorocarbonyls to hydroxycarbonyls (2, 14, 31). The interest in halohydrin dehalogenases increased when i...

The genes involved in isoprene (2-methyl-1,3-butadiene) utilization in Rhodococcus sp. strain AD45 were cloned and characterized. Sequence analysis of an 8.5-kb DNA fragment showed the presence of 10 genes of which 2 encoded enzymes which were previously found to be involved in isoprene degradation: a glutathione S-transferase with activity towards 1,2-epoxy-2-methyl-3-butene (isoI) and a 1-hydroxy-2-glutathionyl-2-methyl-3-butene dehydrogenase (isoH). Furthermore, a gene encoding a second glutathione S-transferase was identified (isoJ). The isoJ gene was overexpressed in Escherichia coli and was found to have activity with 1-chloro-2,4-dinitrobenzene and 3,4-dichloro-1-nitrobenzene but not with 1,2-epoxy-2-methyl-3-butene. Downstream of isoJ, six genes (isoABCDEF) were found; these genes encoded a putative alkene monooxygenase that showed high similarity to components of the alkene monooxygenase from Xanthobacter sp. strain Py2 and other multicomponent monooxygenases. The deduced amino acid sequence encoded by an additional gene (isoG) showed significant similarity with that of ␣-methylacyl-coenzyme A racemase. The results are in agreement with a catabolic route for isoprene involving epoxidation by a monooxygenase, conjugation to glutathione, and oxidation of the hydroxyl group to a carboxylate. Metabolism may proceed by fatty acid oxidation after removal of glutathione by a still-unknown mechanism.2-Methyl-1,3-butadiene (isoprene) is a volatile compound that is emitted in large quantities from plants, especially under thermal stress conditions. The annual global emission from plants is estimated to be 500 million tons, which is similar to the global emission of methane (35). In addition, isoprene emission from several bacteria has been detected (50). Isoprene plays an important role in atmospheric chemistry since it is involved in the generation of ozone and carbon monoxide (42). Despite this, little work on the microbial degradation of isoprene has been done. Cleveland and Yavitt (8) showed that microorganisms consume isoprene even when it is present at trace level concentrations and that soil microorganisms may provide a significant biological sink for atmospheric isoprene. Information about the physiology of isoprene degradation is scarce, however. van Ginkel et al. (44) have shown that degradation in a Nocardia sp. starts with a monooxygenase which oxidizes isoprene to 1,2-epoxy-2-methyl-3-butene and 1,2-3,4-diepoxybutane. Ewers and Knackmuss (13) reported the presence of a glutathione-dependent activity towards 1,2-epoxy-2-methyl-3-butene in cell extracts of an isoprene-utilizing Rhodococcus sp., but the enzyme catalyzing this reaction and the products formed were not further characterized. Moreover, no work on the genetics of isoprene metabolism has been published.In our laboratory, Rhodococcus sp. strain AD45 was isolated for its capability to use isoprene as the sole source of carbon and energy. The pathway of isoprene degradation in strain AD45 starts with oxidation by a monooxygenase to yield 1,2-...

[figure: see text] The halohydrin dehalogenase from Agrobacterium radiobacter AD1 catalyzed the highly enantioselective and beta-regioselective azidolysis of (substituted) styrene oxides. By means of kinetic resolutions the remaining epoxide and the formed azido alcohol could be obtained in very high ee. In a large scale conversion, the decrease in yield and selectivity due to the uncatalyzed chemical side reaction could be overcome by slow addition of azide.

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