Abstract:Sterol 14-demethylase P450 (CYP51) is an essential enzyme for sterol biosynthesis by eukaryotes. We have cloned rat and human CYP51 cDNAs [Aoyama, Y., Noshiro, M., Gotoh, O., Imaoka, S., Funae, Y., Kurosawa, N., Horiuchi, T., and Yoshida, Y. (1996) J. Biochem. 119, 926-933]. The cloned rat CYP51 cDNA was expressed in Escherichia coli with modification of the N-terminal amino acid sequence, and the expressed protein (CYP51m) was purified to gel-electrophoretic homogenity. The spectrophotometrically determined s… Show more
“…The positional specific incorporation of deuterium during catalytic isomerization proceeds as predicted by the mechanism to C-9α [1]. From the limited information on catalytic-centre activities of pure enzymes that act on sterols k cat data are reported for human sterol 8-isomerase 0.423 s −" (this study) and for 14αdemethylase from rat is 0.24 s −" [30]. The activity of pure enzymes from the lanosterol-ergosterol pathway of S. cere isiae act even slower than the enzymes from the cholesterol pathway with catalytic-centre activities for the sterol methyl transferase of 0.01 s −" [18] and for 14α-demethylase of 0.003 s −" [31].…”
CHO 2, encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg 2 yeast cells and purified to apparent homogeneity. The enzyme exhibited a K (m) of 50 microM and a turnover number of 0.423 s(-1) for zymosterol, an isoelectric point of 7.70, a native molecular mass of 107000 Da and was tetrameric. The structural features of zymosterol provided optimal substrate acceptability. Biomimetic studies of acid-catalysed isomerization of zymosterol resulted in formation of cholest-8(14)-enol, whereas the enzyme-generated product was a Delta(7)-sterol, suggesting absolute stereochemical control of the reaction by hSI. Using (2)H(2)O and either zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GC-MS of the deuterated products. The positional specific incorporation of deuterium at C-9alpha was established by a combination of (1)H- and (13)C-NMR analyses of the enzyme-generated cholesta-7,24-dienol. Kinetic analyses indicated the reaction equilibrium ( K (eq)=14; DeltaG(o')=-6.5 kJ/mol) for double-bond isomerization favoured the forward direction, Delta(8) to Delta(7). Treatment of hSI with different high-energy intermediate analogues produced the following dissociation constants ( K (i)): emopamil (2 microM)=tamoxifen (1 microM)=tridemorph (1 microM)<25-azacholesterol (21 microM)
“…The positional specific incorporation of deuterium during catalytic isomerization proceeds as predicted by the mechanism to C-9α [1]. From the limited information on catalytic-centre activities of pure enzymes that act on sterols k cat data are reported for human sterol 8-isomerase 0.423 s −" (this study) and for 14αdemethylase from rat is 0.24 s −" [30]. The activity of pure enzymes from the lanosterol-ergosterol pathway of S. cere isiae act even slower than the enzymes from the cholesterol pathway with catalytic-centre activities for the sterol methyl transferase of 0.01 s −" [18] and for 14α-demethylase of 0.003 s −" [31].…”
CHO 2, encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg 2 yeast cells and purified to apparent homogeneity. The enzyme exhibited a K (m) of 50 microM and a turnover number of 0.423 s(-1) for zymosterol, an isoelectric point of 7.70, a native molecular mass of 107000 Da and was tetrameric. The structural features of zymosterol provided optimal substrate acceptability. Biomimetic studies of acid-catalysed isomerization of zymosterol resulted in formation of cholest-8(14)-enol, whereas the enzyme-generated product was a Delta(7)-sterol, suggesting absolute stereochemical control of the reaction by hSI. Using (2)H(2)O and either zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GC-MS of the deuterated products. The positional specific incorporation of deuterium at C-9alpha was established by a combination of (1)H- and (13)C-NMR analyses of the enzyme-generated cholesta-7,24-dienol. Kinetic analyses indicated the reaction equilibrium ( K (eq)=14; DeltaG(o')=-6.5 kJ/mol) for double-bond isomerization favoured the forward direction, Delta(8) to Delta(7). Treatment of hSI with different high-energy intermediate analogues produced the following dissociation constants ( K (i)): emopamil (2 microM)=tamoxifen (1 microM)=tridemorph (1 microM)<25-azacholesterol (21 microM)
“…The 10 conserved amino acids throughout the whole CYP51 family in the B‘ helix/BC loop and helices F and G are seen in the alignment in Figure . 1 Sequence alignment of 44 CYP51 family members from different biological kingdoms [bacteria ( − ), mycetozoa (), plants ( − ), fungi ( − ), and animals ( − )] in the regions of the B‘ helix/BC loop and helices F and G. The sequences were taken from NCBA, SWISS-PROT, TrEMBL, and TIGR databases. The alignment was performed using Clustal W1.81 and prepared in ESPript 2.0 programs.…”
CYP51 (sterol 14 alpha-demethylase) is an essential enzyme in sterol biosynthetic pathways and the only P450 gene family having catalytically identical orthologues in different biological kingdoms. The proteins have low sequence similarity across phyla, and the whole family contains about 40 completely conserved amino acid residues. Fifteen of these residues lie in the secondary structural elements predicted to form potential substrate recognition sites within the P450 structural fold. The role of 10 of these residues, in the B' helix/BC loop, helices F and G, has been studied by site-directed mutagenesis using as a template the soluble sterol 14 alpha-demethylase of known structure, CYP51 from Mycobacterium tuberculosis (MT) and the human orthologue. Single amino acid substitutions of seven residues (Y76, F83, G84, D90, L172, G175, and R194) result in loss of the ability of the mutant MTCYP51 to metabolize lanosterol. Residual activity of D195A is very low, V87A is not expressed as a P450, and A197G has almost 1 order of magnitude increased activity. After purification, all of the mutants show normal spectral properties, heme incorporation, and the ability to be reduced enzymatically and to interact with azole inhibitors. Profound influence on the catalytic activity correlates well with the spectral response to substrate binding, effect of substrate stabilization on the reduced state of the P450, and substrate-enhanced efficiency of enzymatic reduction. Mutagenesis of corresponding residues in human CYP51 implies that the conserved amino acids might be essential for the evolutionary conservation of sterol 14 alpha-demethylation from bacteria to mammals.
“…The lanosterol 14-demethylase activity of F1 or F2 was assayed according to our previous method. 16,20) The reaction mixture (1.0 ml) contained F1 or F2 corresponding to 0.01 nmol CYP51 and 0.24 unit of the reductase, 15 nmol lanosterol dispersed with 0.04 mg of dilauroylphosphatidylcholine (DLPC), 80 mM potassium phosphate buffer, pH 7.5 containing 0.05% sodium cholate, and 0.15 mM NADPH with its generator (glucose 6-phosphate and glucose-6-phosphate dehydrogenase). The reaction was carried out aerobically at 30°C for 15-20 min.…”
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...
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