Han Qian, Gao Yi Gui, Robinson Howard, Ding Haizhen, Wilson Scott, Li Jianyong
Department of Pathobiology, University of Illinois, Urbana, IL 61802, USA.
FEBS J. 2005 May;272(9):2198-206. doi: 10.1111/j.1742-4658.2005.04643.x.
Aedes aegypti kynurenine aminotransferase (AeKAT) catalyzes the irreversible transamination of kynurenine to kynurenic acid, the natural antagonist of NMDA and 7-nicotinic acetycholine receptors. Here, we report the crystal structure of AeKAT in its PMP and PLP forms at 1.90 and 1.55 A, respectively. The structure was solved by a combination of single-wavelength anomalous dispersion and molecular replacement approaches. The initial search model in the molecular replacement method was built with the result of single-wavelength anomalous dispersion data from the Br-AeKAT crystal in combination with homology modeling. The solved structure shows that the enzyme is a homodimer, and that the two subunits are stabilized by a number of hydrogen bonds, salts bridges, and hydrophobic interactions. Each subunit is divided into an N-terminal arm and small and large domains. Based on its folding, the enzyme belongs to the prototypical fold type, aminotransferase subgroup I. The three-dimensional structure shows a strictly conserved 'PLP-phosphate binding cup' featuring PLP-dependent enzymes. The interaction between Cys284 (A) and Cys284 (B) is unique in AeKAT, which might explain the cysteine effect of AeKAT activity. Further mutation experiments of this residue are needed to eventually understand the mechanism of the enzyme modulation by cysteine.
埃及伊蚊犬尿氨酸转氨酶（AeKAT）催化犬尿氨酸不可逆地转氨生成犬尿酸，犬尿酸是N-甲基-D-天冬氨酸（NMDA）和7-烟碱型乙酰胆碱受体的天然拮抗剂。在此，我们分别报道了AeKAT处于磷酸吡哆醛（PMP）和磷酸吡哆醛（PLP）形式时，分辨率为1.90 Å和1.55 Å的晶体结构。该结构通过单波长反常色散和分子置换方法相结合得以解析。分子置换方法中的初始搜索模型是结合来自溴化AeKAT晶体的单波长反常色散数据结果与同源建模构建而成的。解析出的结构表明该酶是一个同型二聚体，并且两个亚基通过大量氢键、盐桥和疏水相互作用得以稳定。每个亚基分为一个N端臂以及小结构域和大结构域。基于其折叠方式，该酶属于原型折叠类型，即转氨酶亚组I。三维结构显示出一个具有PLP依赖性酶特征的严格保守的“PLP - 磷酸结合杯”。Cys284（A）和Cys284（B）之间的相互作用在AeKAT中是独特的，这可能解释了AeKAT活性的半胱氨酸效应。需要对该残基进行进一步的突变实验，以最终了解半胱氨酸对该酶的调节机制。
