RRC ID 54407
Author Kitahama Y, Nakamura M, Yoshida Y, Aoyama Y.
Title The construction and characterization of self-sufficient lanosterol 14-demethylase fusion proteins consisting of yeast CYP51 and its reductase.
Journal Biol. Pharm. Bull.
Abstract Two forms of a self-sufficient lanosterol 14-demethylase fused enzyme consisting of Saccharomyces cerevisiae CYP51 and S. cerevisiae reduced nicotinamide-adenine dinucleotide phospahte (NADPH)-P450 reductase were constructed and characterized. The two forms of fused enzymes, F1 and F2, which had slight differences in the linker regions between their P450 and reductase domains, were expressed in Escherichia coli cells. Both F1 and F2 were purified to homogeneity. The purified preparations of F1 and F2 showed spectral properties of not only P450 but also flavoprotein. F1 and F2 showed lanosterol 14-demethylase activity with kinetic parameters comparable to those obtained with a reconstituted system consisting of S. cerevisiae CYP51 and S. cerevisiae NADPH-P450 reductase. These facts indicate that F1 and F2 are self-sufficient lanosterol 14-demethylases that can catalyze three successive monooxygenations with comparable activity to naturally occurring CYP51. The enzymatic reduction of the CYP51 in F1 and F2 was faster than that of the CYP51 in the reconstituted system. The results of dilution experiments suggested that the electron transfer from the reductase domain to the CYP51 domain in F1 and F2 occurred both intra- and intermolecularly. Two fused self-sufficient lanosterol 14-demethylases were successfully constructed. This is the first example of the purified preparation of an artificial self-sufficient P450 monooxygenase that catalyzes the oxidative cleavage of C-C bond via three successive monooxygenations.
Volume 32(4)
Pages 558-63
Published 2009-4
PII JST.JSTAGE/bpb/32.558
PMID 19336884
MeSH Catalysis Cell Membrane / enzymology Cytochrome P-450 Enzyme System / biosynthesis* Cytochrome P-450 Enzyme System / chemistry Cytochrome P-450 Enzyme System / genetics* DNA, Complementary / biosynthesis DNA, Complementary / genetics DNA, Fungal / biosynthesis DNA, Fungal / genetics Electron Transport Escherichia coli / genetics NADPH-Ferrihemoprotein Reductase / biosynthesis* NADPH-Ferrihemoprotein Reductase / chemistry NADPH-Ferrihemoprotein Reductase / genetics* Recombinant Fusion Proteins / biosynthesis Recombinant Fusion Proteins / chemistry Recombinant Fusion Proteins / genetics Saccharomyces cerevisiae / chemistry Saccharomyces cerevisiae / enzymology* Saccharomyces cerevisiae / genetics* Sterol 14-Demethylase
IF 1.694
Resource
DNA material Yeast CYP51F1(erg11)m/pBKS- (RDB16756) Yeast NADPH-P450 reductase/pBKS- (RDB16760)