Crystal Structure and Computational Analyses Provide Insights into the Catalytic Mechanism of 2,4-Diacetylphloroglucinol Hydrolase PhlG from Pseudomonas fluorescens
Crystal Structure and Computational Analyses Provide Insights into the Catalytic Mechanism of 2,4-Diacetylphloroglucinol Hydrolase PhlG from Pseudomonas fluorescens
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DOI：
10.1074/jbc.m109.044180
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发表时间：
2010-02-12
影响因子：
4.8
通讯作者：
Zhou, Cong-Zhao
中科院分区：
文献类型：
--
作者：
He, Yong-Xing;Huang, Liang;Zhou, Cong-Zhao
来自荧光假单胞菌的2，4-二乙酰基间苯三酚水解酶PhlG催化抗生素2，4-二乙酰基间苯三酚的水解碳-碳（C-C）键裂解以形成单乙酰基间苯三酚，这是化学和生物化学中一类罕见的反应。为了研究这种酶的催化机制，我们使用X射线晶体学和MAD方法在2.0埃分辨率下确定了PhlG的三维结构。PhlG的整体结构包括一个主要参与二聚化的小N端结构域和一个Bet v1样折叠的C端结构域，这将PhlG与经典的α/β折叠水解酶区分开来。哑铃形的基板通道被确定为连接一个狭窄的内部两亲性口袋的外部溶剂。在底物与活性位点结合后，通道可能发生显著的构象变化。结合计算对接研究、定点诱变和酶活性分析的结构分析表明，2，4-二乙酰基间苯三酚C-C键的裂解通过水分子的亲核攻击进行，水分子由锌离子协调。此外，残基Tyr（121）、Tyr（229）和Asn（132），其被预测为与羟基基团和未水解的乙酰基基团氢键结合，可以精细地调节并将结合的底物定位在反应性取向中。这些结果揭示了PhlG的活性中心和锌依赖性水解机制，并解释了其底物特异性。
2,4-Diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens catalyzes hydrolytic carbon-carbon (C-C) bond cleavage of the antibiotic 2,4-diacetylphloroglucinol to form monoacetylphloroglucinol, a rare class of reactions in chemistry and biochemistry. To investigate the catalytic mechanism of this enzyme, we determined the three-dimensional structure of PhlG at 2.0 angstrom resolution using x-ray crystallography and MAD methods. The overall structure includes a small N-terminal domain mainly involved in dimerization and a C-terminal domain of Bet v1-like fold, which distinguishes PhlG from the classical alpha/beta-fold hydrolases. A dumbbell-shaped substrate access tunnel was identified to connect a narrow interior amphiphilic pocket to the exterior solvent. The tunnel is likely to undergo a significant conformational change upon substrate binding to the active site. Structural analysis coupled with computational docking studies, site-directed mutagenesis, and enzyme activity analysis revealed that cleavage of the 2,4-diacetylphloroglucinol C-C bond proceeds via nucleophilic attack by a water molecule, which is coordinated by a zinc ion. In addition, residues Tyr(121), Tyr(229), and Asn(132), which are predicted to be hydrogen-bonded to the hydroxyl groups and unhydrolyzed acetyl group, can finely tune and position the bound substrate in a reactive orientation. Taken together, these results revealed the active sites and zinc-dependent hydrolytic mechanism of PhlG and explained its substrate specificity as well.