Gustafsson Mattias C U, Roitel Olivier, Marshall Ker R, Noble Michael A, Chapman Stephen K, Pessegueiro Antonio, Fulco Armand J, Cheesman Myles R, von Wachenfeldt Claes, Munro Andrew W
Department of Cell and Organism Biology, Lund University, Sölvegatan 35, SE-223, 62 Lund, Sweden.
Biochemistry. 2004 May 11;43(18):5474-87. doi: 10.1021/bi035904m.
The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically. Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding. CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates. Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes. Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate. Both P450s 102A2 and 102A3 show kinetic and binding preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates. P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles. Titration curves for binding and turnover kinetics of several fatty acid substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding. Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B. subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.
编码巨大芽孢杆菌中黄素细胞色素P450 BM3（CYP102A1）的两种枯草芽孢杆菌同源物（CYP102A2和CYP102A3）的cyp102A2和cyp102A3基因已被克隆、在大肠杆菌中表达、纯化，并进行了光谱和酶学表征。两种酶都含有血红素、黄素腺嘌呤二核苷酸（FAD）和黄素单核苷酸（FMN）辅因子，并能结合多种脂肪酸分子，这可通过底物结合诱导的血红素铁的低自旋静止形式向高自旋形式的转变来证明。CYP102A2和CYP102A3能高效催化还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的脂肪酸依赖性氧化以及人工电子受体的还原。一氧化碳与两种酶的还原形式结合会导致血红素Soret带移至450 nm，证实了这些酶的P450性质。对酶与肉豆蔻酸反应产物进行反相高效液相色谱（HPLC）分析表明，两者都能催化该底物的亚末端羟基化反应，不过区域选择性和催化速率不同。P450 102A2和102A3对长链不饱和脂肪酸和支链脂肪酸的动力学和结合偏好均高于饱和脂肪酸，这表明前两种分子类型可能是真正的底物。P450 102A2和102A3彼此之间以及与P450 BM3的底物选择性谱不同，这表明它们可能发挥着略有不同的细胞作用。P450 102A2和102A3与几种脂肪酸底物的结合和周转动力学滴定曲线用S形（而非双曲线）函数能更好地描述，这表明有不止一个底物分子与P450结合，或者可能存在底物结合的协同作用。对这三种黄素细胞色素的氨基酸序列进行比较表明，P450 BM3中的几个重要氨基酸在枯草芽孢杆菌同源物中并不保守，这表明底物结合模式存在差异，这可能解释了异常的S形动力学和滴定特性。
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