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Insights into Sequence–Activity Relationships amongst Baeyer–Villiger Monooxygenases as Revealed by the Intragenomic Complement of Enzymes from Rhodococcus jostii RHA1

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Abstract

TheRhodococcus jostiiRHA1 genome encodes a number of enzymes that can be exploited as biocatalysts. Study of the substrate spectrum and enantioselectivity of Baeyer–Villiger monooxygenases from R. jostii allowed the identification of short amino acid sequences specific to groups displaying certain catalytic characteristics. The gel illustrates the substrate acceptance spectra and selectivities of the different proteins.

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Microbial genome sequences are providing a wealth of information on new enzymes that have considerable potential as biocatalysts. The recently sequenced genome of Rhodococcus jostii RHA1, for example, has revealed an impressive array of catabolic enzymes, including many putative Baeyer–Villiger monooxygenases (BVMOs). We have cloned 23 target BVMO sequences from the genome of R. jostii RHA1 and heterologously expressed 13 of these as soluble proteins to unearth new substrate specificities and selectivities. Whole-cell biocatalysts expressing the genes were screened against seven different test substrates. Each of these catalysts displayed activity toward at least three ketones. We observed a remarkable diversity of both regio- and enantioselectivity among the BVMOs from R. jostii RHA1 for the transformation of two chiral substrates, with some enzymes displaying high enantioselectivity for the isomers of 2-methylcyclopentanone. With the notable exception of the product of gene ro03437, named MO14, the biocatalysts' sequences correlated well with their respective activities and selectivities. This correlation allowed the identification of sequence motifs specific to subgroups of the BVMOs from R. jostii and other organisms. Overall, the data improve predictive models of BVMO activity from sequence and suggest new avenues to pursue in engineering these enzymes.

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