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

为治理环境污染，必须尽快开发降解有害有机卤化物的脱卤酶。本研究从原子水平对假单胞菌降解有机卤化物底物的机理进行了研究。YL L-2-卤酸脱卤酶，采用蛋白质工程技术。天然酶的2*X-射线晶体结构分析表明，该二聚体酶的每个亚基由两个结构域组成：核心区具有活性中心的α/β结构，四股线束结构域用于二聚。随后，将S175A突变体晶体浸泡在不同底物L-2-卤酸溶液中制备的配合物的2*晶体结构揭示了侧链羧氧与底物C2原子共价键合的酯中间体的结构。在中间体中，Asp10-Thr14区域向活性中心移动，底物失去卤素，其构型从L构型变为D-构型。此外，在每一种情况下，都在Ser175羟基附近发现了一个新的水分子。对于底物L-2-卤代酰胺，电子密度峰位于Arg41的侧链附近，可能参与了从底物中提取卤素的过程。在每个中间体中，底物的羧基通过与Ser118羟基和Asn119的主链氨基的氢键稳定，底物的烷基通过疏水口袋中的疏水作用稳定。

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.