Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Patent
1998-01-27
2000-08-08
Witz, Jean C.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
435 691, 435132, 530402, C12N 902, C12P 700, C12P 2106, C07K 100
Patent
active
061000744
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
Mono-oxygenases catalyse the selective oxidation of non-functionalised hydrocarbons using oxygen.sup.1, and are therefore of great interest for potential use in organic synthesis. However, progress in this area has been hampered by the difficulty in isolating sufficient quantities of enzyme and the associated electron-transfer proteins. Despite the availability of amino acid sequences of more than 150 different cytochrome P-450 mono-oxygenases, to date structural data of only three are available.sup.2,3,4, and few have been successfully over-expressed in bacterial systems.sup.5.
One cytochrome P-450 mono-oxygenase, which is soluble and can be expressed in sufficient quantities, is the highly specific P-450.sub.cam from P.putida which catalyses the regio- and stereo-selective hydroxylation of camphor (1) to 5-exo-hydroxycamphor.sup.6. The high resolution crystal structure of P-450.sub.cam has been determined.sup.2, and since the mechanism of action of this bacterial enzyme is believed to be very similar to that of its mammalian counterparts, it has been used as a framework on which models of mammalian enzymes are based.
The nucleotide sequence and corresponding amino acid sequence of P-450.sub.cam have been described.sup.5. The location of an active site of the enzyme is known and structure-function relationships have been investigated.sup.13, 14. Mutants of P-450.sub.cam have been described, at the 101 and 185 and 247 positions.sup.15, and at the 87 position.sup.16. A mutant in which tyrosine 96 has been changed to phenyl alanine-96 has been described.sup.12,17,18. But in all these cases the papers report effects of the mutations on the mechanisms of known oxidation reactions. There is no teaching or suggestion that mutation might be used to provide biocatalysts for oxidation of different substrates.
SUMMARY OF THE INVENTION
In an attempt to find new biocatalysts, we have initiated a project which aims to redesign the active site of P-450.sub.cam, such that it is able to carry out specific oxidations of organic molecules which are not substrates for the wild-type protein. Our initial aim was to incorporate an "aromatic pocket" into the P-450.sub.cam active site, which would encourage the binding of substrates containing aromatic side-chains.
In addition, a surface residue remote from the active site was identified (cysteine-334) with effects on protein handling and stability. The cysteine is responsible for unwanted dimerisation of the protein during purification and an alanine residue was therefore substituted for the cysteine in order to improve both of these properties.
The three dimensional structure of P-450.sub.cam shows the active site to provide close van der Waals contact with the hydrophobic groups of camphor as shown in FIG. 1. Three aromatic residues (Y96, F87 and F98) are grouped together and line the substrate binding pocket, with a hydrogen bond between tyrosine 96 and the camphor carbonyl oxygen maintaining the substrate in the correct orientation to ensure the regio-and stereo-specificity of the reaction. Replacement of any of these aromatic residues with a smaller, hydrophobic non-aromatic side-chain could provide the desired "aromatic pocket".
Molecular modelling was used to investigate the likely effects of point mutations to the three aromatic residues. The program GRID.sup.7 was used to calculate an energy of interaction between an aromatic probe and possible mutants of cytochrome P-450.sub.cam where these residues were changed to alanine (F87A, Y96A and F98A). The results were then examined graphically using the molecular modelling package Quanta.sup.8.
The mutant F98A appeared to have the strongest binding interaction within the active site cavity accessible to the aromatic probe, with that of Y96A being slightly smaller, and that of F87A being substantially less. It was decided in the first instance to mutate tyrosine 96 to alanine as it is more central to the binding pocket, whereas phenylalanine 98 is in a groove to one side. Also, removal of tyros
REFERENCES:
Richardson et al. "Alterations of the regiospecificity of progesterone metabolism by the mutagenesis of two key amino acid residues in rabbit cytochrome P450 2C3v," J. Biol. Chem. (1994) 269(39): 23937-43.
Di Primo et al. "Mutagenesis of a single hydrogen bond in cytochrome P450 alters cation binding and heme solvation," J. Biol. Chem. (1990) 265(10): 5361-63.
Atkins et al. "The roles of active site hydrogen bonding in cytochrome P450cam as revealed by site-directed mutagenesis," J. Biol. Chem. (1988) 263(35): 18842-49.
William M. Atkins and Stephan G. Sligar, "Molecular Recognition in Cytochrome P-450: Alteration of Regioselective Alkane Hydroxylation via Protein Engineering," J. Am. Chem. Soc., 1989, 111(7). 2715-2717.
Flitsch Sabine Lahja
Nickerson Darren Paul
Wong Luet-Lok
BG plc
Hanley Susan
Holt William H.
Witz Jean C.
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