PURIFICATION AND CHARACTERIZATION OF CYTOCHROME-P450(RR1) FROM RHODOCOCCUS-RHODOCHROUS
PURIFICATION AND CHARACTERIZATION OF CYTOCHROME-P450(RR1) FROM RHODOCOCCUS-RHODOCHROUS
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DOI：
10.1111/j.1432-1033.1993.tb17750.x
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发表时间：
1993-04-01
期刊：
影响因子：
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通讯作者：
TIMMIS, KN
中科院分区：
文献类型：
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作者：
ELTIS, LD;KARLSON, U;TIMMIS, KN
可溶性细胞色素P450的合成诱导的2-乙氧基苯酚，并结合纯化至表观同质性从紫红红球菌菌株116。该酶的亚基分子量为44.5 kDa，通过SDS/PAGE测定，pI为5.2。电子吸收光谱表明，在不存在底物的情况下，天然细胞色素主要处于低自旋状态（在50 mM Mops，pH 7.0，25 ℃中为13%高自旋状态）。2-甲氧基苯酚以0.53 +/- 0.03 μ M（50 mM Mops，pH 7.0，25 ℃）的宏观解离常数与细胞色素结合，并诱导血红素铁99.7%转变为五配位高自旋形式。使用重构的体外活性测定，证明P450 RR 1催化2-乙氧基苯酚和2-甲氧基苯酚的O-脱烷基化以产生邻苯二酚。细胞色素结合其他邻位取代的酚类，包括2-乙氧基苯酚、2-甲基苯酚（邻甲酚）和2-氯苯酚。P450 RR 1对这些化合物的亲和力低于2-甲氧基苯酚，并且它们在诱导血红素铁向高自旋态转变方面不如2-甲氧基苯酚有效。对位取代和间位取代的醚酚没有诱导自旋跃迁。
A soluble cytochrome P450 whose synthesis is induced by and that binds 2-ethoxyphenol was purified to apparent homogeneity from Rhodococcus rhodochrous strain 116. The enzyme had a subunit molecular mass of 44.5 kDa as determined by SDS/PAGE and a pI of 5.2. The electronic absorption spectrum indicates that the native cytochrome in the absence of substrate is predominantly in the low-spin state (13% high-spin state in 50 mM Mops, pH 7.0, 25-degrees-C). 2-Methoxyphenol binds to the cytochrome with a macroscopic dissociation constant of 0.53 +/- 0.03 muM (50 mM Mops, pH 7.0, 25-degrees-C) and induces a 99.7% transition of the heme iron to the pentacoordinate high-spin form. Using a reconstituted in-vitro activity assay, it was demonstrated that P450RR1 catalyzed the O-dealkylation of 2-ethoxyphenol and 2-methoxyphenol to produce catechol. The cytochrome binds other ortho-substituted phenols, including 2-ethoxyphenol, 2-methylphenol (o-cresol) and 2-chlorophenol. The affinity of P450RR1 for these compounds is lower than that of 2-methoxyphenol and they are less effective than 2-methoxyphenol at inducing a transition in the heme iron to the high-spin state. Para-substituted and meta-substituted ether phenols did not induce a spin transition.