Recently, a protein sequence motif has been identified which facilitates identification of putative BVMO genes [11]. This enables effective screening of the available (genome) sequence databases. We have surveyed the presently available sequenced microbia genomes (607) which yielded just over 400 putative type I BVMO sequences. This number is in sharp contrast with the limited number of BVMOs available in recombinant form. It is also striking to note that all identified genes originate from bacteria and fungi.

Figure 3.4 shows all known type I BVMO sequences together with the type I BVMO sequences obtained from fully sequenced microbial genomes and the metagenome sequence database in a dendrogram representation. This illustrates the clustering of several sets of sequences in a restricted number of clades. Clustering of several known BVMOs with similar substrate specificities in specific clades suggests that these groups represent BVMOs that act on similar substrates. For example, cyclododecanone monooxygenase (CDDMO) [12] and cyclopentadecanone monooxygenase (CPDMO) [13] have overlapping substrate specificities and are clustered in a distinct clade of 16 BVMO sequences. All these BVMOs probably represent monooxygenases acting on bulky cyclic aliphatic ketones. As can be seen in Fig. 3.4, a similar situation is seen for cyclopentanone monooxygenases (CPMOs) [14] and most cyclohexanone monooxygenases [1517], which form two separate clades. This is in agreement with the observation that CPMOs and CHMOs often display different regio- and enantioselectivities [18, 19].

The clustering of enzymes with similar substrate specificities suggests that sequence analysis can be used as a predictive tool to obtain hints concerning substrate specificities of uncharacterized BVMOs. However, most clades contain only one or even no BVMO sequence of which the biocatalytic properties are known. The clade in which HAPMO [20] is located is quite dispersed and totally new substrate specificities can be expected from sequences that are part of this group of BVMO sequences. There is also a distinct cluster of putative BVMO sequences that entails ethionamide monooxygenase from Mycobacterium tuberculosis [21]. It has been shown that this BVMO is able to catalyze a range of Baeyer-Villiger reactions accepting both aromatic and aliphatic substrates. However, the physiological substrate of this enzyme is as yet unknown.

Comamonas Testosteroni Information

Fig. 3.4 An unrooted phylogenetic tree of type I BVMO protein sequences. Sequences were retrieved from the NCBI and PEDANT sequence databases. In addition to known BVMOs and putative BVMOs identified in annotated microbial genomes, putative homologs retrieved from the metagenome sequence database of the Sargasso sea (NCBI) are also indicated (dotted lines). All characterized BVMOs and their physiological substrates are indicated: CDDMO, cyclododecanone monooxygenase from Rhodococcus rhodochrous; CPDMO, cyclopentadecanone monooxygenase from Pseudomonas HI-70; CPMO1, cyclopentanone monooxygenase from Comamonas testosteroni; CPMO2, cyclopentanone monooxygenases from Brevibacterium sp. HCU; CHMO1, cyclohexanone monooxygenase from Acinetobacter NCIMB

9871; CHMO2, cyclohexanone monooxygenase from Arthrobacter sp. BP2; CHMO3 and CHMO4, cyclohexanone monooxygenases from Rhodococcus sp. Phi1 and Phi2, respectively; CHMO5, cyclohexanone monooxygenase from Xanthobacter autotrophicus Py2; CHMO6, cyclohexanone monooxygenases from Brevibacterium sp. HCU; STMO, steroid monooxygenase from Rhodococcus rhodochrous; PAMO, phenylacetone monooxygenase from Thermobifida fusca; HAPMO, 4-hydroxyacetophenone monooxygenase from Pseudomonasfluorescens ACB; EtaA, ethionamide-activating monooxygenase from Mycobacterium tuberculosis H37Rv. Several BVMOs that are discussed in the text as "unexplored BVMOs" and Mtub5 are also indicated.

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