Probing the interactions of putidaredoxin with redox partners in camphor p450 5-monooxygenase by mutagenesis of surface residues
Probing the interactions of putidaredoxin with redox partners in camphor p450 5-monooxygenase by mutagenesis of surface residues
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DOI：
10.1074/jbc.272.35.21720
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发表时间：
1997-08-29
影响因子：
4.8
通讯作者：
Vilker, V
中科院分区：
文献类型：
--
作者：
Holden, M;Mayhew, M;Vilker, V
通过定点诱变研究了表面氨基酸残基在恶臭氧还蛋白 (Pdx) 与其细胞色素 P450(cam) (CYP101) 系统中的氧化还原伙伴相互作用中的作用。突变的Pdx基因在大肠杆菌中表达，并在体外纯化和研究蛋白质。测量了完整重构的 P450(cam) 系统的活性，并确定了动力学参数。还进行了部分测定以确定突变对与每个氧化还原伙伴相互作用的影响。一些突变改变了 Pdx 与一个氧化还原配偶体的相互作用，但不改变另一个氧化还原配偶体的相互作用，其他突变影响了与两个氧化还原配偶体的相互作用，表明 Pdx 上的恶臭氧还蛋白还原酶和 CYP101 的结合位点有一些重叠。 Pdx 的半胱氨酸 73 被认为在 Pdx 与恶臭氧还蛋白还原酶的相互作用中非常重要，而天冬氨酸 38 在亚基结合和电子转移到 CYP101 中起着关键作用。
The role of surface amino acid residues in the interaction of putidaredoxin (Pdx) with its redox partners in the cytochrome P450(cam) (CYP101) system was investigated by site-directed mutagenesis. The mutated Pdx genes were expressed in Escherichia coli, and the proteins were purified and studied in vitro. Activity of the complete reconstituted P450(cam) system was measured, and kinetic parameters were determined, Partial assays were also conducted to determine the effect of the mutations on interactions with each redox partner. Some mutations altered interactions of Pdx with one redox partner but not the other, Other mutations affected interactions with both redox partners, suggesting some overlap in the binding sites on Pdx for putidaredoxin reductase and CYP101. Cysteine 73 of Pdx was identified as important in the interaction of Pdx with putidaredoxin reductase, whereas aspartate 38 serves a critical role in the subunit binding and electron transfer to CYP101.