Award details

Construction of a biotechnologically versatile and stable oxygenase biocatalyst

Reference	BB/C006879/2
Principal Investigator / Supervisor	Professor Andrew Munro
Co-Investigators / Co-Supervisors	Dr Hazel Girvan, Professor David Leys, Professor Nigel Scrutton
Institution	The University of Manchester
Department	Chem Eng and Analytical Science
Funding type	Research
Value (£)	193,248
Status	Completed
Type	Research Grant
Start date	01/09/2005
End date	31/07/2008
Duration	35 months

Abstract

The use of enzymes (engineered and otherwise) in the chemical and biotechnology industries is desirable in view of their relative efficiency in generating specific and chiral products, and since much of the dirty and frequently toxic waste products from traditional organic synthetic chemistry processes can be avoided. However, factors mitigating against greater exploitation of enzymes in industry are frequently those that revolve around their instability and tendency to undergo denaturation (with loss of structure and activity) under conditions required for the industrial transformation and/or with prolonged incubation for maximal product yield. Engineering of enzymes (by both rational and forced evolution methods) provides a means by which they can be endowed with physical and catalytic properties that could allow them to be more useful biocatalysts in industrial scenarios. Redox enzymes are attracting increasing interest as biotechnologically useful catalysts, e.g. for production of high value pharmaceuticals such as polyketide antibiotics and e.g. for generation of chiral compounds that bypass the requirements for extensive clean up processes to remove the unwanted enantiomers. A leading class of enzymes that lend themselves well to requirements of the chemical and biotechnological industries are the cytochromes P450 (P450s), which catalyse stereo- and regio-selective oxygenation of a wide range of organic molecules using molecular oxygen as the substrate and electrons delivered from NADPH via an exogenous redox partner. The flavocytochrome P450 BM3 enzyme from Bacillus megaterium has features that make it a system of choice for biotechnological applications and exploitation. It is a single component redox system with a FAD- and FMN-containing NADPH-dependent P450 reductase fused to a fatty acid hydroxylase P450 in a single polypeptide. The fusion arrangement facilitates extremely rapid electron transfer through the enzyme, resulting P450 BM3 being the highest activity oxygenase in the entire P450 superfamily of enzymes to date. Moreover, structural studies of the enzyme are well advanced and the structures of substrate-free and substrate-bound forms are known. In addition, extensive rational mutagenesis and forced evolution studies have produced a series of mutants of varying substrate selectivity and capable of performing reactions of interest in the industrial sector for production of high value molecules of use as pharmaceuticals and chiral synthons. As with other P450s, the factors that currently mitigate against the use of P450 BM3 in the biotech sector are its relative instability in prolonged use (particularly relating to loss of catalytically relevant heme ligation, dissociation of heme and FMN cofactors from the protein matrix, and general structural instability of the heme- and FMN-binding domains) and its preference for NADPH, which is a much more expensive reducing coenzyme than NADH. In this proposal we week to address all of these weaknesses in BM3 through a systematic programme of rational mutagenesis. Previous studies of related family 4 P450s show that the heme macrocycle can be covalently linked to the protein and we will engineer this feature into BM3 to avoid loss of heme ligation (P420 formation) and dissociation of the heme. Weak FMN binding is an obvious consequence of the absence of stabilising interactions with the cofactor that are observed in flavodoxins and other related proteins. We will mutagenise to introduce these favourable interactions, and will also engineer surface disulfides at key points on the surface of the FMN and heme domains of the enzyme to further strengthen the enzyme. Finally, we will generate primary mutants to facilitate coenzyme switch from NADPH to NADH, and secondary mutations in the coenzyme-binding site to prevent inhibition of the enzyme by oxidised forms of these coenzymes (NAD(P)). Mutant enzymes will be thoroughly characterised using an array of kinetic, thermodynamic, structural and biophysical techniques. Beneficial mutations will be combined and robustness of the mutants will be determined in stability assays. The capacity of the mutants to catalyse biotechnologically important reactions will be established, leading to production of a range of more stable and biotechnologically compatible BM3 mutants that are useful in industrial operations.

Summary

unavailable

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	Industrial Biotechnology, Microbiology, Structural Biology, Synthetic 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

Associated awards:

BB/C006879/1 Construction of a biotechnologically versatile and stable oxygenase biocatalyst

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