Unexplored Type I BVMOs

In recent years, several type I BVMO genes have been identified that are associated with various specific microbial anabolic or catabolic pathways. Few biochemical data exist on these enzymes. However, based on the elucidated biosynthesis or degradation pathways, the identity of the physiological substrate of these type I BVMOs can be deduced as will be discussed below.

Fumonisin toxins are a potentially serious problem as they are frequently found as contaminants in maize and maize-based food [65]. These mycotoxins are produced by plant pathogenic fungi, mainly Fusarium species. It has been found that fumonisin Bj can be degraded by the fungus Exophiala spinifera by the combined action of a fumonisin esterase, an oxidase, and a BVMO [65, 66]. The BVMO is held responsible for removing the aldehyde tail of a fumonisin derivative (Fig. 3.5). Application of these enzymes would reduce the health risks associated with


Fig. 3.5 Structural formulas of BVMO substrates. Arrows indicate the carbon-carbon bond that is cleaved upon Baeyer-Villiger oxidation.


Fig. 3.5 Structural formulas of BVMO substrates. Arrows indicate the carbon-carbon bond that is cleaved upon Baeyer-Villiger oxidation.

maize-fed livestock. As can be seen in Fig. 3.4 ("Fum" sequence), a comparison of the respective protein sequence with other BVMOs reveals that cyclopentanone monooxygenases are the closest homologs (36% seq. ident.). This indicates that modest changes in protein sequence can result in a significantly altered substrate acceptance profile as fumonisin derivatives substantially deviate in structure from cyclopentanone.

Two other recently reported type I BVMO sequences also show relatively high sequence identity with cyclopentanone monooxygenases and have been identified in another fungus, Neotyphodium uncinatum [67]. This grass-endophytic fungus is capable of producing unusual alkaloids, loline alkaloids, which protect the host plants from insects. Two orthologous gene clusters were identified in the genome of this fungus that entail copies of the genes necessary to build the loline alkaloids. Both gene clusters contain a cyclopentanone monooxygenase homolog ("LolF" sequence, see Fig. 3.4). Based on the proposed biosynthetic route to nor-loline, it is reasonable to assume that these BVMOs are involved in the defor-mylation of a proline derivative as indicated in Fig. 3.5. This again shows the widely varying substrate specificities of microbial BVMOs.

Another fungal type I BVMO that is involved in the biosynthesis of a secondary metabolite catalyzes a crucial step towards aflatoxin biosynthesis in aspergilli. Disruption of the respective BVMO gene in Aspergillus parasiticus, moxY, results in accumulation of aflatoxin precursors [68]. The aflatoxin precursor that normally undergoes a Baeyer-Villiger reaction is indicated in Fig. 3.5. The MoxY BVMO and its orthologs are only distantly related in sequence to well-characterized type I BVMOs (see "MoxY" in Fig. 3.4).

In 2000 it was shown that a type I BVMO in Mycobacterium tuberculosis is solely responsible for the activation of antitubercular thioamides into bactericidal products (Fig. 3.5) [69, 70]. Mutations in the respective etaA gene (gene Rv3854c) result in multidrug resistance towards these thioamide antibiotics. Studies in our laboratory have revealed that EtaA represents a true type I BVMO. We have shown that EtaA can convert a number of substrates via a Baeyer-Villiger reaction [21]. Interestingly, one of the substrates (benzylacetone) of EtaA identified in our study has recently been shown to be of potential use in treating tuberculosis. By applying ethionamide in combination with this EtaA substrate, Mycobacterium tuberculosis became more sensitive to ethionamide [71]. This unforeseen synergistic effect opens new avenues for the development of a more effective tuberculosis treatment based on the action of EtaA.

It has been shown that EtaA is active with a range of thioamides and ketones, providing no clear clue concerning the real physiological substrate. The observed promiscuity in substrate recognition is in line with the broad substrate acceptance of other type I BVMOs. Sequence comparison with other type I BVMOs shows that EtaA belongs to a defined group of BVMOs that are only distantly related to other characterized BVMOs (Fig. 3.4).

Future studies will provide more insight into the substrate specificity of the abovementioned BVMOs. As shown in the previous section, type I BVMOs usually accept a much broader range of compounds than their physiological substrates. For example, it has been shown that CHMO accepts well over 100 different non-physiological substrates, mainly cyclic aliphatic ketones. Newly identified BVMOs might facilitate biocatalysis with a broader range of compounds, including substrates containing many functional groups that are structurally unrelated to current CHMO substrates.

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