NRC Publications Record / Notice d'Archives des publications de CNRC:http://nparc.cisti-icist.nrc-cnrc.gc.ca/npsi/ctrl?action=rtdoc&an=21275605&lang=en http://nparc.cisti-icist.nrc-cnrc.gc.ca/npsi/ctrl?action=rtdoc&an=21275605&lang=fr READ THESE TERMS AND CONDITIONS CAREFULLY BEFORE USING THIS WEBSITE.http://nparc.cisti-icist.nrc-cnrc.gc.ca/npsi/jsp/nparc_cp.jsp?lang=en Vous avez des questions? Nous pouvons vous aider. Pour communiquer directement avec un auteur, consultez la première page de la revue dans laquelle son article a été publié afin de trouver ses coordonnées. Si vous n'arrivez pas à les repérer, communiquez avec nous à PublicationsArchive-ArchivesPublications@nrc-cnrc.gc.ca. Questions? Contact the NRC Publications Archive team atPublicationsArchive-ArchivesPublications@nrc-cnrc.gc.ca. If you wish to email the authors directly, please see the first page of the publication for their contact information. NRC Publications Archive Archives des publications du CNRCThis publication could be one of several versions: author's original, accepted manuscript or the publisher's version. / La version de cette publication peut être l'une des suivantes : la version prépublication de l'auteur, la version acceptée du manuscrit ou la version de l'éditeur. For the publisher's version, please access the DOI link below./ Pour consulter la version de l'éditeur, utilisez le lien DOI ci-dessous.http://doi.org/10.1021/cb500442e Chemical Biology, 9, 12, pp. 2843-2851, 2014 Lactone ABSTRACT: The Baeyer−Villiger monooxygenases (BVMOs) are microbial enzymes that catalyze the synthetically useful Baeyer−Villiger oxidation reaction. The available BVMO crystal structures all lack a substrate or product bound in a position that would determine the substrate specificity and stereospecificity of the enzyme. Here, we report two crystal structures of cyclohexanone monooxygenase (CHMO) with its product, ε-caprolactone, bound: the CHMO Tight and CHMO Loose structures. The CHMO Tight structure represents the enzyme state in which substrate acceptance and stereospecificity is determined, providing a foundation for engineering BVMOs with altered substrate spectra and/or stereospecificity. The CHMO Loose structure is the first structure where the product is solvent accessible. This structure represents the enzyme state upon binding and release of the substrate and product. In addition, the role of the invariant Arg329 in chaperoning the substrate/product during the catalytic cycle is highlighted. Overall, these data provide a structural framework for the engineering of BVMOs with altered substrate spectra and/or stereospecificity. ACS

The Baeyer-Villiger monooxygenases (BVMOs) are a group of bacterial enzymes that are able to catalyze the synthetically useful Baeyer-Villiger oxidation reaction. As such, these enzymes have attracted considerable attention as potential industrial biocatalysts. The interest in these enzymes has led to a desire to be able to rationally design them for tailored biocatalytic applications. While recent years have seen the publication of a number of crystal structures (1-3), we have been lacking a structure of a BVMO that has its native substrate or product bound in a conformation that will allow the determination of substrate specificity and stereospecificity. Without such a structure, progress towards tailored BVMOs has been hampered. We have been able to solve two crystal structures of cyclohexanone monooxygenase (CHMO) with its lactone product, ε-caprolactone, bound. These structures place the lactone in an ideal position for the determination of its substrate specificity and stereospecificity. These structures have provided us with a better understanding of the structural basis for substrate binding, paving the way for the rational design of tailored BVMOs. At the same time, we have pursued small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) studies to better understand the dynamic nature of the enzyme. These studies have allowed us to explain the relationship between the various crystallized states of BVMOs and their complex, fourteen step enzyme mechanism.