Ileperuma Nadeesha R, Marshall Sean D G, Squire Christopher J, Baker Heather M, Oakeshott John G, Russell Robyn J, Plummer Kim M, Newcomb Richard D, Baker Edward N
School of Biological Sciences, University of Auckland, Auckland, New Zealand.
Biochemistry. 2007 Feb 20;46(7):1851-9. doi: 10.1021/bi062046w. Epub 2007 Jan 26.
Carboxylesterases (CXEs) are widely distributed in plants, where they have been implicated in roles that include plant defense, plant development, and secondary metabolism. We have cloned, overexpressed, purified, and crystallized a carboxylesterase from the kiwifruit species Actinidia eriantha (AeCXE1). The structure of AeCXE1 was determined by X-ray crystallography at 1.4 A resolution. The crystal structure revealed that AeCXE1 is a member of the alpha/beta-hydrolase fold superfamily, most closely related structurally to the hormone-sensitive lipase subgroup. The active site of the enzyme, located in an 11 A deep hydrophobic gorge, contains the conserved catalytic triad residues Ser169, Asp276, and His306. Kinetic analysis using artificial ester substrates showed that the enzyme can hydrolyze a range of carboxylester substrates with acyl groups ranging from C2 to C16, with a preference for butyryl moieties. This preference was supported by the discovery of a three-carbon acyl adduct bound to the active site Ser169 in the native structure. AeCXE1 was also found to be inhibited by organophosphates, with paraoxon (IC50 = 1.1 muM) a more potent inhibitor than dimethylchlorophosphate (DMCP; IC50 = 9.2 muM). The structure of AeCXE1 with paraoxon bound was determined at 2.3 A resolution and revealed that the inhibitor binds covalently to the catalytic serine residue, with virtually no change in the structure of the enzyme. The structural information for AeCXE1 provides a basis for addressing the wider functional roles of carboxylesterases in plants.
羧酸酯酶(CXEs)广泛分布于植物中,在植物防御、植物发育和次生代谢等多种作用中发挥功能。我们从猕猴桃属植物毛花猕猴桃(AeCXE1)中克隆、过量表达、纯化并结晶了一种羧酸酯酶。通过X射线晶体学以1.4埃的分辨率确定了AeCXE1的结构。晶体结构显示,AeCXE1是α/β-水解酶折叠超家族的成员,在结构上与激素敏感脂肪酶亚组关系最为密切。该酶的活性位点位于一个11埃深的疏水峡谷中,包含保守的催化三联体残基Ser169、Asp276和His306。使用人工酯底物进行的动力学分析表明,该酶能够水解一系列酰基范围从C2到C16的羧酸酯底物,对丁酰部分具有偏好性。在天然结构中发现与活性位点Ser169结合的三碳酰基加合物,支持了这种偏好性。还发现AeCXE1受到有机磷酸盐的抑制,对氧磷(IC50 = 1.1 μM)比二甲基氯磷酸酯(DMCP;IC50 = 9.2 μM)是更强效的抑制剂。以2.3埃的分辨率确定了与对氧磷结合的AeCXE1的结构,结果显示抑制剂与催化丝氨酸残基共价结合,而酶的结构几乎没有变化。AeCXE1的结构信息为研究羧酸酯酶在植物中更广泛的功能作用提供了基础。