The effect of mutation of F87 on the properties of CYP102A1-CYP4C7 chimeras: altered regiospecificity and substrate selectivity.
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 omega-1, omega-2, and omega-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.
Chen
Chiung-Kuang J
CK
Department of Chemistry, University of Arizona, Tucson, AZ, 85721-0041, USA.
Shokhireva
Tatiana Kh
TKh
Berry
Robert E
RE
Zhang
Hongjun
H
Walker
F Ann
FA
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics*; D017931:DNA Primers; D005204:Farnesol / Q000378:metabolism; D007850:Lauric Acids / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D050506:Mutant Chimeric Proteins / Q000737:chemistry* / Q000235:genetics*; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics*; D010084:Oxidation-Reduction; D019308:Palmitic Acid / Q000378:metabolism; D017354:Point Mutation / Q000502:physiology*; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013054:Spectrophotometry, Atomic; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D007850:Lauric Acids; D050506:Mutant Chimeric Proteins; C030358:lauric acid; D019308:Palmitic Acid; D005204:Farnesol; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases