Mutant mono-oxygenase cytochrome P450cam

Details Mutant mono-oxygenase cytochrome P450cam Mutant mono-oxygenase cytochrome P450cam

1998-09-14

2000-09-12

Achutamurthy, Ponnathapu

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

435 691, 4352523, 4353201, 435471, 536 232, C12N 902, C12N 1500, C12N 1553, C12N 1578

Patent

active

061176613

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to a mutant of the mono-oxygenase cytochrome P-450cam.


BACKGROUND OF THE INVENTION

Mono-oxygenases catalyse the selective oxidation of activated and unactivated carbon-hydrogen bonds 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 the mono-oxygenase enzyme and/or 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 date 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-450cam from P. putida which catalyses the regio- and stereo-selective hydroxylation of camphor to 5-exo-hydroxycamphor.sup.6. The high resolution crystal structure of P-450cam 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 structural models of mammalian enzymes are based.
The nucleotide sequence and corresponding amino acid sequence of P-450cam have been described.sup.5.7. The location of an active site of the enzyme is known and structure-function relationships have been investigated.sup.8.9. Mutants of P-450cam have been described at the 101 and 185 and 247 and 295 positions.sup.9.10.11. and at the 87 position.sup.12. A mutant in which tyrosine 96 (Y96) has been changed to phenylalanine 96 (the Y96F mutant) has been described.sup.11.13.14.15.. But in all cases the papers report effects of the mutations on the oxidation reactions of molecules which had previously been shown to be substrates for the wild-type enzyme. There is no teaching of how mutations might be used to provide biocatalysts for oxidation of different, novel substrates.


SUMMARY OF THE INVENTION

In an attempt to develop new biocatalysts, we have initiated a project which aims to redesign P-450cam, such that it is able more effectively to carry out specific oxidations of organic molecules whether or not these are substrates for the wild-type protein.


BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that the three dimensional structure of P-450cam shows the active site to provide close van der Waals contact with the hydrophobic groups of camphor.
FIG. 2A and FIG. 2B are gas chromatographs in accord with the present invention.
The three dimensional structure of P-450cam shows the active site to provide close van der Waals contacts with the hydrophobic groups of camphor as shown in FIG. 1. Of particular significance are the contacts between camphor and the side chains of leucine 244, valine 247 and valine 295. 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.
Lipscomb and co-workers.sup.16 demonstrated in 1978 that wild-type P-450cam showed a propensity to dimerise, but they also reported that the catalytic activity of the monomer and dimer towards camphor oxidation were indistinguishable. Since the dimerisation reaction could be reversed by thiol reducing agents, they concluded that it occurred by intermolecular cysteine disulphide (S--S) bond formation. They were unable to determine whether dimerisation involved more than one cysteine per P-450cam molecule. Nor were they able to identify the key cysteine residue(s) involved in this reaction because neither the amino acid sequence nor crystal structure of P-450cam were known at the time.
We used molecular modelling to investigate the likely effects of points mutations to the three aromatic residues (Y96, F87, F98) in the active site pocket. We noted th

REFERENCES:
Smith, C. A. D., et al., 1992, "Debrisoquine hydroxylase gene polymorphism and susceptibilty to Parkinson's disease", The Lancet, vol. 339, pp. 1375-1377.
Tuck, S. F., et al., 1993, "Active sites of the cytochrome p450cam (CYP101) F87W and F87A mutants", The Journal of Biological Chemistry, vol. 268, pp. 269-275.
Dawson, E., et al., 1994, "An association study of debrisoquine hydroxylase (CYP2D6) polymorphisms in schizophrenia", Psychiatric Genetics, vol. 4, pp. 215-218.
J. Biological Chemistry, Dec. 15, 1988, vol. 263 No.35, pp. 18842-18849; William M. Atkins et al., "The Roles of Active Site Hydrogen Bonding in Cytochrome P450cam as Revealed by Site-directed Mutagenesis."
J. AM. Chem. Soc., 1989, vol 111, No. 7, pp. 2715-2717; William M. Atkins et al., "Molecular Recognition in Cytochrome P-450: Alteration of Regioselective Alkane Hydroxylation via Protein Engineering."
J. Biological Chemistry, Apr. 5, 1990, vol. 265, No. 10, pp. 5361-5363; Carmelo Di Primo et al., "Mutagenesis of a Single Hydrogen Bond in Cytochrome P-450 Alters Cation Binding and Heme Solvation."

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