Structure of Escherichia coli glutamate decarboxylase (GADα) in complex with glutarate at 2.05 Å resolution
Structure of Escherichia coli glutamate decarboxylase (GADα) in complex with glutarate at 2.05 Å resolution
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DOI：
10.1107/s0907444904032147
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发表时间：
2005-03-01
影响因子：
2.2
通讯作者：
Sukhareva, BS
中科院分区：
文献类型：
--
作者：
Dutyshev, DI;Darii, EL;Sukhareva, BS
谷氨酸脱羧酶（GAD）是一种吡哆醛酶，催化L-谷氨酸转化为γ-氨基丁酸和二氧化碳。大肠杆菌酶以两种同工酶存在，称为GAD α和GAD β。重组同工酶GAD α与作为底物类似物的复合物的晶体在空间群R3中生长，晶胞参数a = B = 117.1，c = 196.4埃。酶的结构通过分子置换法解析，并在2.05埃分辨率下精确到15.1%的R因子（无R = 19.9%）。不对称单位包含一个二聚体，由两个亚基组成的酶相关的非晶体学的二重轴，这是垂直于和相交的晶体学的三重轴。二聚体通过晶体学三重轴相关以形成六聚体。每个亚基的活性位点由二聚体的两个亚基的大结构域的残基形成。辅酶磷酸吡哆醛（PLP）与Lys 276形成醛亚胺键。结合在酶活性中心的戊二酸分子采用两种占据率相等的构象。戊二酸的两个羧基之一在两种构象中占据相同的位置，并与二聚体的一个亚基的Phe 63的主链和Thr 62的侧链的N原子以及相邻亚基的Asp 86和Asn 83的侧链形成氢键。显然，正是在这个位置，底物的远端羧基将被酶结合，从而提供酶对谷氨酸的识别。
Glutamate decarboxylase (GAD) is a pyridoxal enzyme that catalyzes the conversion Of L-glutamate into gamma-aminobutyric acid and carbon dioxide. The Escherichia coli enzyme exists as two isozymes, referred to as GADalpha and GADbeta Crystals of the complex of the recombinant isozyme GADa with glutarate as a substrate analogue were grown in space group R3, with unit-cell parameters a = b = 117.1, c = 196.4 Angstrom. The structure of the enzyme was solved by the molecular-replacement method and refined at 2.05 Angstrom resolution to an R factor of 15.1% (R-free = 19.9%). The asymmetric unit contains a dimer consisting of two subunits of the enzyme related by a noncrystallographic twofold axis which is perpendicular to and intersects a crystallographic threefold axis. The dimers are related by a crystallographic threefold axis to form a hexamer. The active site of each subunit is formed by residues of the large domains of both subunits of the dimer. The coenzyme pyridoxal phosphate (PLP) forms an aldimine bond with Lys276. The glutarate molecule bound in the active site of the enzyme adopts two conformations with equal occupancies. One of the two carboxy groups of the glutarate occupies the same position in both conformations and forms hydrogen bonds with the N atom of the main chain of Phe63 and the side chain of Thr62 of one subunit and the side chains of Asp86 and Asn83 of the adjacent subunit of the dimer. Apparently, it is in this position that the distal carboxy group of the substrate would be bound by the enzyme, thus providing recognition of glutamic acid by the enzyme.