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Baeyer-Villiger enzymes and application in biotransformations

Reference	BB/D004403/1
Principal Investigator / Supervisor	Professor Jennifer Littlechild
Co-Investigators / Co-Supervisors
Institution	University of Exeter
Department	Biosciences
Funding type	Research
Value (£)	202,240
Status	Completed
Type	Research Grant
Start date	02/11/2005
End date	01/07/2009
Duration	44 months

Abstract

This proposal aims to determine the structural basis of the stereoselectivity of enzyme catalysed Baeyer-Villiger reactions. The type 1 and type 2 enzymes are unrelated in sequence and subunit content. We propose to capitalise on our recent structural determination of the type 2 DKMO 3,6 diketocamphane enzyme oxygenating subunit and to locate the flavin and substrate binding sites in this enzyme. The involvement of specific amino acids in the enzymatic mechanism will be investigated by site-directed mutagenesis. We will attempt to solve the structure of the complex of this enzyme with its dehydrogenase subunit. We will also continue with our studies on a type 1 enzymes - the MO2 camphor pathway enzyme and collaborate with Dr Fraaije on the recently solved structure for the phenylacetone monooxygenase and its similarity to MO2. Once structural information is available this will be used to understand the enzymatic mechanism and rationalise the well established substrate specificity of the two different BVMO enzymes. Site-directed mutagenesis will be carried out to further optimise the enzymes for commercial biocatalytic purposes. These enzymes have traditionally been hard to purify in any reasonable quantities from their host wild type cells, Pseudomonas putida and Acinetobacter sp. With sequence information now available from the large CAM plasmid from P. putida and the cloning of the Acinetobacter BVMO we can extend our studies on the structure and mechanism of this important group of enzymes.

Summary

The proposed research will provide information about an important group of monoxygenase enzymes that have relevance for biocatalytic processes important for UK industry. They represent a novel group of flavoenzymes that are able to carry out a so called Baeyer-Villiger reaction where dioxygen and another NAD(P)H cofactor are used to covalently introduce an oxygen into a range of organic molecules. They fall into two discrete groups where the type 1 enzymes contain both cofactors on single polypeptide chain and the type 2 enzymes are multi subunit with a dimeric oxygenating subunit and a separate dehydrogenase component. We have recently solved the structure of the oxygenating subunit of the type 2 enzyme which is shown to be related to bacterial luciferase and an unusual archaeal dehydrogenase. We now want to extend these studies to locate the flavin and substrate binding sites in this enzyme and to crystallise the complex with its dehydrogenase. We will also embark on a crystallographic study of a type 1 enzyme. This work should be facilitated now that the genes coding for these enzymes have been located on the CAM plasmid from Pseudomonas species. This will allow sufficient amounts of protein to be obtained. Once the structural information is available we will be able rationalise a wealth of substrate specificity data available from previously carried out biotransformation studies. We will also be in a position to use site-directed mutagenesis to broaden the substrates that can be used by these enzymes.

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	Industrial Biotechnology, Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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