Mechanism of enhanced conversion of 1,2,3-trichloropropane by mutant haloalkane dehalogenase revealed by molecular modeling
Mechanism of enhanced conversion of 1,2,3-trichloropropane by mutant haloalkane dehalogenase revealed by molecular modeling
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DOI：
10.1007/s10822-006-9071-1
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发表时间：
2006-06-01
影响因子：
3.5
通讯作者：
Damborsky, Jiri
中科院分区：
文献类型：
--
作者：
Banas, Pavel;Otyepka, Michal;Damborsky, Jiri
1，2，3-三氯丙烷（TCP）是环氧氯丙烷生产过程中的一种高毒性、难降解的副产品。来自红球菌属（Rhodococcus sp.）的卤代烷烃脱卤酶（DhaA）水解包括TCP在内的各种卤代化合物中的碳-卤键，但效率较低（k（cat）/K-m = 36 s（-1）M（-1））。一个Cys 176 Tyr-DhaA突变体具有三倍更高的催化效率TCP脱卤先前已获得易错PCR。我们已经使用分子模拟和量子力学计算来阐明参与改进的突变体的催化作用，和对映选择性的DhaA对TCP的分子机制。Cys 176 Tyr突变改变蛋白质进入和输出途径。用体积较大的Tyr取代Cys残基使上部通道变窄，使得第二通道“槽”成为优选的路线。TCP在DhaA酶中可以采用两种主要取向，其中一种是卤素稳定残基Asn 41与TCP分子的末端卤素原子形成氢键，而另一种是与中心卤素原子键合。这些结合模式的差异解释了在酶催化的反应中优先形成2，3-二氯丙烷-1-醇的（R）-对映体而不是（S）-对映体。
1,2,3-Trichloropropane (TCP) is a highly toxic, recalcitrant byproduct of epichlorohydrin manufacture. Haloalkane dehalogenase (DhaA) from Rhodococcus sp. hydrolyses the carbon-halogen bond in various halogenated compounds including TCP, but with low efficiency (k(cat)/K-m = 36 s(-1)M(-1)). A Cys176Tyr-DhaA mutant with a threefold higher catalytic efficiency for TCP dehalogenation has been previously obtained by error-prone PCR. We have used molecular simulations and quantum mechanical calculations to elucidate the molecular mechanisms involved in the improved catalysis of the mutant, and enantioselectivity of DhaA toward TCP. The Cys176Tyr mutation modifies the protein access and export routes. Substitution of the Cys residue by the bulkier Tyr narrows the upper tunnel, making the second tunnel "slot" the preferred route. TCP can adopt two major orientations in the DhaA enzyme, in one of which the halide-stabilizing residue Asn41 forms a hydrogen bond with the terminal halogen atom of the TCP molecule, while in the other it bonds with the central halogen atom. The differences in these binding patterns explain the preferential formation of the (R)- over the (S)-enantiomer of 2,3-dichloropropane-1-ol in the reaction catalyzed by the enzyme.