Abstract
The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily.
Highlights
The use of enzymes in commercial biocatalysis is of increasing importance for the fine-chemical and pharmaceutical industries, with the number of new drugs produced in this way due to increase dramatically over the few years (Wohlgemuth, 2010; Bornscheuer et al, 2012; Reetz, 2013)
The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer– Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Aresolution
The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model
Summary
The use of enzymes in commercial biocatalysis is of increasing importance for the fine-chemical and pharmaceutical industries, with the number of new drugs produced in this way due to increase dramatically over the few years (Wohlgemuth, 2010; Bornscheuer et al, 2012; Reetz, 2013). The substrate specificity of type II BVMOs is different from the type I enzymes and they are increasingly important for commercial biocatalysis since they have the ability to oxidize bicyclic lactones (Alphand et al, 2003) and because they utilize the cheaper cofactor NADH It has been known for some time that the bacterium Pseudomonas putida NCIMB 10007 is able to grow on either enantiomer of the natural bicyclic monoterpene camphor as the sole carbon source (LeGall et al, 1963). This has allowed the location of the bound cofactor and a comparison between the native enzyme and cofactorcomplex structures to be carried out
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