The Construction and Characterization of Self-Sufficient Lanosterol 14-Demethylase Fusion Proteins Consisting of Yeast CYP51 and Its Reductase

Abstract

Cytochrome P450 (P450) is a group of heme-protein monooxygenases that catalyze the oxidation of a wide variety of organic compounds including steroids, fatty acids, xenobiotics, and so forth. P450 requires two electrons to activate one oxygen molecule in its catalytic cycle, and the electrons are provided from reduced nicotinamide-adenine dinucleotide phospahte (NADPH) or reduced nicotinamide-adenine dinucleotide (NADH) by means of a specific electrontransferring system, such as NADPH-P450 reductase (P450 reductase), cytochrome b 5 plus NADH-cytochrome b 5 reductase, or iron-sulfur protein plus its reductase. 1) Although most P450 and electron-transferring proteins are independent proteins encoded by different genes, there are a few self-sufficient monooxygenases that contain both P450 and P450 reductase in a single polypeptide chain. A typical example of such a self-sufficient P450 monooxygenase is the long-chain fatty acid hydroxylase P450 BM-3 of Bacillus megaterium. 2)This monooxygenase consists of P450 (CYP102A1) and P450 reductase domains and shows extremely high molecular turnover (Ͼ1500/min).3) This high activity is considered to be due to efficient intramolecular electron transfer from the reductase domain to the P450 domain. This has prompted many P450 investigators to construct recombinant self-sufficient drug and steroid metabolizing P450s using various mammalian P450 species and P450 reductase, and many recombinant self-sufficient P450 monooxygenases have been reported. [4][5][6][7][8][9][10][11][12] These studies indicate that a self-sufficient artificial P450 monooxygenase can be constructed by joining P450 and P450 reductase by protein engineering.Some specific P450 species such as CYP19 and CYP51 catalyze oxidative cleavage of C-C bond through three successive monooxygenation reactions, 13) and it would be interesting to construct a self-sufficient C-C bond-cleaving P450. Sterol 14-demethylase P450 (CYP51) is a unique P450 that is widely distributed among five biological kingdoms as a conserved P450 species, 14) and a fusion protein consisting of CYP51 and an iron-sulfur protein that displays sterol 14-demethylase activity in the presence of ferredoxin reductase has been found in Methylococcus capsulatus.15) However, the electron transfer system to eukaryotic CYP51 is different. Eukaryotic CYP51 receives electrons from P450 reductase, although the bacterial enzyme receives electrons via an electron-transfer system consisting of an iron-sulfur protein and a flavoprotein. The authors will attempt to construct a selfsufficient lanosterol 14-demethylase fusion protein by using yeast CYP51 and P450 reductase. This paper reports the construction of two kinds of recombinant self-sufficient lanosterol 14-demethylase and their molecular and catalytic properties. 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. Th...

Novel Substrate Specificity and Temperature-Sensitive Activity of Mycosphaerella graminicola CYP51 Supported by the Native NADPH Cytochrome P450 Reductase

“…The modeling experiments suggested that lanosterol could fit into the active site, as supported by the type I binding spectra, but could not orientate viously calculated K s value of 11 M for eburicol binding to MgCYP51 by UV-visible spectroscopy (16) suggesting that the K m value is not solely dependent on the affinity of MgCYP51 for substrate. The MgCYP51 K m value for eburicol was comparable with that determined for human CYP51 of 30 M with lanosterol (30) but was 4-to 6-fold larger than K m values previously determined for C. albicans and S. cerevisiae CYP51 enzymes (15,31,32), suggesting a degree of variability with regard to substrate affinity among CYP51 enzymes.…”

Expression, Purification, and Characterization of Aspergillus fumigatus Sterol 14-α Demethylase (CYP51) Isoenzymes A and B

“…The absolute spectra of AF51B in the presence and absence of 70 M lanosterol indicated that a less than 1% change in spin state had occurred. The K s value obtained for lanosterol was comparable to the K m value of 7.4 M for lanosterol reported for C. albicans CYP51 in metabolism studies (39) but 2-fold higher than the K m value for S. cerevisiae CYP51 (20). Further investigations are required to fully characterize the substrate binding specificity of AF51B.…”

Interaction between the reductase Tah18 and highly conserved Fe‐S containing Dre2 C‐terminus is essential for yeast viability

“…For the same purpose, Ncp1 and its redox partner Erg11, a lanosterol 14‐alpha‐demethylase that belongs to the cytochrome P450 family (Aoyama et al ., 1996), were fused as a single protein. The enzymatic reduction of Erg11 was faster in the purified fusion protein than in the reconstituted complex, due to electron transfer occurring both inter‐ and intra‐molecularly (Kitahama et al ., 2009).…”