细胞色素P450BM3进化酶3D结构模建与分析

Modeling of 3D Structure of Cytochrome P450BM3 Evolved Enzyme

通过动力学参数测定、同源序列比较以及三维结构的模建,对三个能够羟基化吲哚生成靛蓝的高活力P450BM3进化酶E435T,D168H,D168NA225VK440N所导致的酶分子结构变化和可能的活力提高机制进行了初步分析。同源序列比较表明P450BM3晶体蛋白的氨基酸序列与同源的能够羟基化吲哚生成靛蓝的动物P450等同率只有13%～16%,并且这些突变位点均在同源的P450中没有发现。三维结构模建表明进化酶E435T高活力可能是该突变破坏了E435侧链羧基和K434赖氨酸侧链胍基之间强的氢键相互作用,使K434胍基这个带正电荷基团侧链的柔韧性增加;而进化酶D168H高活力可能是通过影响P450BM3空间结构来影响酶催化活性的改变。

Kinetic parameters, three dimensional structures and homogenous sequence alignment of evolved P450 BM3 E435T,D168H,D168NA225VK440N that can hydroxylate indole into indigo were compared to illustrate the molecular structural changes and mechanisms that improve catalytic properties. Sequence identities of P450BM3 and other homogenous P450s are generally low （13%-16%）, and none of its mutations can be found in the other homogenous P450s. From the modeling, it can be supposed that glutamic acid substituted by threonine at position 435 breaks the hydrogen bond between carboxyl of side-chain residue at E435 and guanidyl of side-chain residue at K434, which could improve the flexibility of side chain of lysine at 434 and then influence the P450BM3 activity toward indole. The positive effect of 168 position may be due to a long-range electrostatic interaction on the structure of the protein or to the effect on a conformational change of certain molecular interior. All the comparison results of the evolved P450BM3 provided the basis for the relationship of structure and function.