Three-dimensional structure of Escherichia coli asparagine synthetase B:: A short journey from substrate to product
Three-dimensional structure of Escherichia coli asparagine synthetase B:: A short journey from substrate to product
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DOI：
10.1021/bi9915768
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发表时间：
1999-12-07
期刊：
影响因子：
2.9
通讯作者：
Rayment, I
中科院分区：
文献类型：
--
作者：
Larsen, TM;Boehlein, SK;Rayment, I
天冬酰胺合成酶 B 催化天冬氨酸、Mg(2+)ATP 和谷氨酰胺组装成天冬酰胺。在这里，我们描述了大肠杆菌酶的三维结构，并确定并精化至 2.0 埃分辨率。本研究使用的蛋白质是定点突变蛋白 Cys1Ala。大晶体在谷氨酰胺和 AMP 存在的情况下生长，二聚体蛋白的每个亚基折叠成两个不同的结构域。 N 末端区域包含两层反向平行 β 片层，每层包含六条链。楔入这些片层之间的是负责谷氨酰胺水解的活性位点。用于将谷氨酰胺底物定位在结合袋内的关键侧链包括 Arg 49、Asn 74、Glu 76 和 Asp 98。C 端结构域负责 Mg(2+)ATP 和天冬氨酸的结合，由两侧为 α 螺旋的五链平行 β 折叠主导。 AMP 部分通过与 Ser 346 的 O-gamma 以及 Val 272、Leu 232 和 Gly 347 的骨架羰基和酰胺基的氢键锚定到蛋白质上。正如对其他酰胺转移酶所观察到的，两个活性位点通过主要由骨架原子和疏水性和非极性氨基酸残基排列的隧道连接。引人注目的是，天冬酰胺合成酶 B 的 N 端结构域的三维结构与在谷氨酰胺磷酸核糖焦磷酸酰胺转移酶中观察到的相似，而 C 结构域的分子基序则与在 GMP 合成酶中观察到的相似。
Asparagine synthetase B catalyzes the assembly of asparagine from aspartate, Mg(2+)ATP, and glutamine. Here, we describe the three-dimensional structure of the enzyme from Escherichia coli determined and refined to 2.0 Angstrom resolution. Protein employed for this study was that of a site-directed mutant protein, Cys1Ala. Large crystals were grown in the presence of both glutamine and AMP, Each subunit of the dimeric protein folds into two distinct domains. The N-terminal region contains two layers of antiparallel beta-sheet with each layer containing six strands. Wedged between these layers of sheet is the active site responsible for the hydrolysis of glutamine. Key side chains employed for positioning the glutamine substrate within the binding pocket include Arg 49, Asn 74, Glu 76, and Asp 98. The C-terminal domain, responsible for the binding of both Mg(2+)ATP and aspartate, is dominated by a five-stranded parallel beta-sheet flanked on either side by alpha-helices. The AMP moiety is anchored to the protein via hydrogen bonds with O-gamma of Ser 346 and the backbone carbonyl and amide groups of Val 272, Leu 232, and Gly 347. As observed for other amidotransferases, the two active sites are connected by a tunnel lined primarily with backbone atoms and hydrophobic and nonpolar amino acid residues. Strikingly, the three-dimensional architecture of the N-terminal domain of asparagine synthetase B is similar to that observed for glutamine phosphoribosylpyrophosphate amidotransferase while the molecular motif of the C-domain is reminiscent to that observed for GMP synthetase.