Shi Dashuang, Gallegos Rene, DePonte Joseph, Morizono Hiroki, Yu Xiaolin, Allewell Norma M, Malamy Michael, Tuchman Mendel
Children's Research Institute, Children's National Medical Center, 111 Michigan Avenue N.W., Washington, DC 20010-2970, USA.
J Mol Biol. 2002 Jul 19;320(4):899-908. doi: 10.1016/S0022-2836(02)00539-9.
A transcarbamylase-like protein essential for arginine biosynthesis in the anaerobic bacterium Bacteroides fragilis has been purified and crystallized in space group P4(3)2(1)2 (a=b=153.4 A, c=94.8 A). The structure was solved using a single isomorphous replacement with anomalous scattering (SIRAS) and was refined at 2.0 A resolution to an R-factor of 20.6% (R-free=25.2%). The molecular model is trimeric and comprises 960 amino acid residues, two phosphate groups and 422 water molecules. The monomer has the consensus transcarbamylase fold with two structural domains linked by two long interdomain helices: the putative carbamoyl phosphate-binding domain and a binding domain for the second substrate. Each domain has a central parallel beta-sheet surrounded by alpha-helices and loops with alpha/beta topology. The putative carbamoyl phosphate-binding site is similar to those in ornithine transcarbamylases (OTCases) and aspartate transcarbamylases (ATCases); however, the second substrate-binding site is strikingly different. This site has several insertions and deletions, and residues critical to substrate binding and catalysis in other known transcarbamylases are not conserved. The three-dimensional structure and the fact that this protein is essential for arginine biosynthesis suggest strongly that it is a new member of the transcarbamylase family. A similar protein has been found in Xylella fastidiosa, a bacterium that infects grapes, citrus and other plants.
一种对脆弱拟杆菌中精氨酸生物合成至关重要的转氨甲酰酶样蛋白已被纯化，并在空间群P4(3)2(1)2（a = b = 153.4 Å，c = 94.8 Å）中结晶。该结构通过单对映体置换并结合反常散射（SIRAS）解析得到，并在2.0 Å分辨率下精修至R因子为20.6%（R自由 = 25.2%）。分子模型为三聚体，由960个氨基酸残基、两个磷酸基团和422个水分子组成。单体具有公认的转氨甲酰酶折叠结构，有两个结构域通过两个长的结构域间螺旋相连：假定的氨甲酰磷酸结合结构域和第二个底物的结合结构域。每个结构域都有一个由α螺旋和具有α/β拓扑结构的环包围的中央平行β折叠片层。假定的氨甲酰磷酸结合位点与鸟氨酸转氨甲酰酶（OTCases）和天冬氨酸转氨甲酰酶（ATCases）中的位点相似；然而，第二个底物结合位点却显著不同。该位点有几个插入和缺失，并且在其他已知转氨甲酰酶中对底物结合和催化至关重要的残基并不保守。三维结构以及该蛋白对精氨酸生物合成至关重要这一事实强烈表明它是转氨甲酰酶家族的一个新成员。在感染葡萄、柑橘和其他植物的细菌——木质部难养菌中发现了一种类似的蛋白。
