Abstract
CYP102A1 is a highly active water-soluble bacterial monooxygenase that contains both substrate-binding heme and diflavin reductase subunits, all in a single polypeptide that has been called a “self-sufficient enzyme.” Several years ago we developed a procedure called “scanning chimeragenesis,” where we focused on residues 73–82 of CYP102A1, which contact approximately 40% of the substrates palmitoleic acid and N-palmitoylglycine [Murataliev et al. (2004) Biochemistry 43:1771–1780]. These residues were replaced with the homologous residues of CYP4C7. In the current work, that study has been expanded to include residue 87. Phenylalanine 87 of wild-type CYP102A1 was replaced with the homologous residue of CYP4C7, leucine, as well as with alanine. The full-sized chimeric proteins C(73–78, F87L), C(73–78, F87A), C(75–80, F87L), C(75–80, F87A), C(78–82, F87L) and C(78–82, F87A) have been purified and characterized. Wild-type CYP102A1 is most active toward fatty acids (both lauric and palmitic acids produce ω-1, ω-2, and ω-3 hydroxylated fatty acids), but it also catalyzes the oxidation of farnesol to three products (2, 3- and 10,11-epoxyfarnesols and 9-hydroxyfarnesol). All of the F87-mutant chimeric proteins show dramatic decreases in activities with the natural CYP102A1 substrates. In contrast, C(78–82, F87A) and C(78–82, F87L) have markedly increased activities with farnesol, with the latter showing a 5.7-fold increase in catalytic activity as compared to wild-type CYP102A1. C(78–82, F87L) produces 10,11-epoxyfarnesol as the single primary metabolite. The results show that chimeragenesis involving only the second half of SRS-1 plus F87 is sufficient to change the substrate selectivity of CYP102A1 from fatty acids to farnesol and to produce a single primary product.
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Abbreviations
- CYP102A1:
-
Cytochrome P450 from Bacillus megaterium, also called P450-BM3
- CYP4C7:
-
A membrane-bound cytochrome P450 from the cockroach, Diploptera punctata
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Acknowledgments
We thank Professor Armand Fulco of the University of California Los Angeles for the original E. coli strain pbsBM-3 with the plasmid carrying the CYP102A1 gene, Professor Paul Ortiz de Montellano for helpful comments, Dr. Markus Knipp for assistance in the modeling of the chimeras, Dr. Marat B. Murataliev (University of Alberta, Edmonton, AB, Canada) for technical advice on protein purification, fatty acid and farnesol metabolite identification, and Mary T. Flores for preparing the NMR figures. This research was supported by the Walker Research Gift Fund.
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Chen, CK.J., Shokhireva, T.K., Berry, R.E. et al. The effect of mutation of F87 on the properties of CYP102A1-CYP4C7 chimeras: altered regiospecificity and substrate selectivity. J Biol Inorg Chem 13, 813–824 (2008). https://doi.org/10.1007/s00775-008-0368-5
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DOI: https://doi.org/10.1007/s00775-008-0368-5