Koo Laura S, Immoos Chad E, Cohen Michael S, Farmer Patrick J, Ortiz de Montellano Paul R
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143-0446, USA.
J Am Chem Soc. 2002 May 22;124(20):5684-91. doi: 10.1021/ja017174g.
CYP119, a cytochrome P450 from a thermophilic organism for which a crystal structure is available, is shown here to hydroxylate lauric acid in a reaction supported by putidaredoxin and putidaredoxin reductase. This fatty acid hydroxylation activity is increased 15-fold by T214V and D77R mutations. The T214V mutation increases the rate by facilitating substrate binding and enhancing the associated spin state change, whereas the D77R mutation improves binding of the heterologous redox partner putidaredoxin to CYP119 and the rate of electron transfer from it to the heme group. A sequence alignment with P450(cam) can, therefore, be used to identify a part of the binding site for putidaredoxin on an unrelated P450 enzyme. This information can be used to engineer by mutagenesis an improved complementarity of the protein-protein interface that results in improved electron transfer from putidaredoxin to the P450 enzyme. As a result, the catalytic activity of the thermo- and barostable CYP119 has been incorporated into a catalytic system that hydroxylates fatty acids.
CYP119是一种来自嗜热生物的细胞色素P450，其晶体结构已知。本文显示，在恶臭假单胞菌铁氧还蛋白和恶臭假单胞菌铁氧还蛋白还原酶支持的反应中，CYP119可使月桂酸发生羟基化。T214V和D77R突变使这种脂肪酸羟基化活性提高了15倍。T214V突变通过促进底物结合和增强相关的自旋态变化来提高反应速率，而D77R突变则改善了异源氧化还原伙伴恶臭假单胞菌铁氧还蛋白与CYP119的结合以及从它到血红素基团的电子转移速率。因此，与P450（cam）的序列比对可用于确定无关P450酶上恶臭假单胞菌铁氧还蛋白结合位点的一部分。该信息可用于通过诱变工程改造蛋白质-蛋白质界面，以改善其互补性，从而提高从恶臭假单胞菌铁氧还蛋白到P450酶的电子转移。结果，热稳定和压力稳定的CYP119的催化活性已被纳入一个使脂肪酸羟基化的催化体系中。
