Structural Studies on Degradation Mechanisn of Organohalides by Dehalogenase Using Protein-Engineering Techniques

必须尽快开发分解有害有机卤化物的脱卤酶，以治理环境污染。本研究利用蛋白质工程技术，在原子水平上对假单胞菌YL -2-卤酸脱卤酶降解底物有机卤化物的机理进行了结构研究。二聚体酶的2 * x射线晶体结构分析表明，二聚体酶的每个亚基由两个结构域组成：具有α / β结构的核心结构域和具有二聚体结构的四螺旋束结构域。随后，将S175A突变体晶体浸泡在各种底物（l -2-卤代酸）溶液中制备的配合物的2 *晶体结构显示了侧链羧基氧与底物的C2原子共价结合的酯中间体结构。在中间体中，Asp10-Thr14区域向活性位点移动，底物失去卤素并将其构型从L型转变为d型。此外，在每一种情况下，在ser75羟基附近都发现了一个新的水分子。对于底物l -2-卤酰胺，在Arg41的侧链附近出现了电子密度峰，这可以被认为是从底物中释放的卤化物离子，这可能与从底物中提取卤素有关。在每个中间体中，底物的羧基被与Ser118羟基和Asn119主链氨基的氢键稳定，而底物的arkyl基被疏水口袋中的疏水相互作用稳定。

Dehalogenases for decomposing harmful organohalides must be developed as soon as possible to clean up environmental pollution. In the present research, structural studies at atomic level on mechanism of degrading substrates organohalides by Pseudomonas sp. YL L-2-haloacid dehalogenase, using protein-engineering techniques. The 2 * X-ray crystal structure analysis of the native enzyme has revealed that each subunit of the dimeric enzyme consists of two domains : the core-domain having a alpha/beta structure with the active site, and four-helix bundle domain for dimerization. Subsequently, 2 * crystal structures of complexes prepared by soaking the S175A mutant crystals in solutions of various substrates, L-2-haloacids, have revealed those of ester intermediates where the side-chain carboxyle oxygen is covalently bonded to the C2 atom of the substrate. In the intermediates, the region of Asp10-Thr14 moves towards the active site, and the substrates lose the halogen and change their configuration from L- to D-forms. In each case, moreover, a new water molecule has been found in the vicinity of the Serl75 hydroxyle. In case of the substrate L-2-haloamide, the electron density peak, which can be assigned as the halide ion released from the substrate, has occurred near the side chain of Arg41 which is expected to be involved in abstraction of the halogen from the substrate. In each intermediate, the carboxyle group of the substrate has been stabilized by hydrogen bonds with the Ser118 hydroxyle and the main-chain amino group of Asn119, and the arkyl group of the substrate has been stabilized byhydrophobic interactions in the hydrophobic pocket.