Elucidation of amino acid residues critical for unique activities of rabbit cytochrome P450 2B5 using hybrid enzymes and reciprocal site-directed mutagenesis with rabbit cytochrome P450 2B4
Elucidation of amino acid residues critical for unique activities of rabbit cytochrome P450 2B5 using hybrid enzymes and reciprocal site-directed mutagenesis with rabbit cytochrome P450 2B4
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DOI：
10.1006/abbi.1996.0127
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发表时间：
1996-03-15
影响因子：
3.9
通讯作者：
Burnett, VL
中科院分区：
文献类型：
--
作者：
Szklarz, GD;He, YQ;Burnett, VL
兔细胞色素P450 2B 5的独特活性的分子基础，与高度相关的兔2B 4相比，进行了研究，使用杂交酶和定点突变。在COS细胞中表达的2B 4 - 2B 5杂交体中观察到的雄烯二酮羟化酶谱的改变表明，关键氨基酸存在于N-末端ApaI片段（密码子1-122）和内部SstI片段（密码子220-393）中。基于这些结果，用其它细胞色素P450 2B获得的数据，以及与Gotoh（1992，J.Biol.Chem.267，83-90）提出的6个底物识别位点的相关性，在114、294、363和367位构建了相互2B 4 - 2B 5突变体。在大肠杆菌中表达野生型和突变型酶，并分析了许多底物的氧化。所有研究的残基被发现是重要的雄烯二酮羟基化的区域和立体特异性，在这些位置的突变也引起孕酮，苄氧基试卤灵，pentoxyresorufin，乙氧基香豆素，和苄非他明的氧化的改变，与依赖于酶，残基和底物的变化的幅度和方向。在两种酶中的残基114和294突变后，始终观察到活性的主要变化，并且其中一些改变在P450 2B 4的3-D模型的帮助下进行解释。例如，在2B 4 Ile-114 --> Phe突变体中，Phe阻止雄烯二酮呈现16 β结合方向，并且还阻碍苄氧基试卤灵的结合，导致活性丧失。相反，Phe-114的存在稳定了雄烯二酮的16 α-结合方向，导致该活性的增加。(C)出版社：Academic Press，Inc.
The molecular basis for the unique activities of rabbit cytochrome P450 2B5, compared with the highly related rabbit 2B4, was investigated using hybrid enzymes and site-directed mutagenesis. Alterations in androstenedione hydroxylase profiles observed with 2B4-2B5 hybrids expressed in COS cells showed that key amino acids are present in both the N-terminal ApaI fragment (codons 1-122) and an internal SstI fragment (codons 220-393). Based on these results, data obtained with other cytochromes P450 2B, and correlation to the six substrate recognition sites proposed by Gotoh (1992, J. Biol. Chem. 267, 83-90), reciprocal 2B4-2B5 mutants were constructed at positions 114, 294, 363, and 367. Wild-type and mutant enzymes were expressed in Escherichia coli, and the oxidation of a number of substrates was analyzed. All residues studied were found to be important for regio- and stereospecificity of androstenedione hydroxylation, Mutations at these positions also caused alterations in the oxidation of progesterone, benzyloxyresorufin, pentoxyresorufin, ethoxycoumarin, and benzphetamine, with the magnitude and direction of the changes dependent upon the enzyme, residue, and substrate. Major changes in activity were consistently observed upon mutation of residues 114 and 294 in both enzymes, and some of these alterations were interpreted with the help of a 3-D model of P450 2B4. For example, in the 2B4 Ile-114 --> Phe mutant, Phe prevents androstenedione from assuming a 16 beta-binding orientation and also hinders binding of benzyloxyresorufin, leading to a loss of activity. Conversely, the presence of Phe-114 stabilizes a 16 alpha-binding orientation of androstenedione, resulting in an increase in this activity. (C) 1996 Academic Press, Inc.