Gulick A M, Palmer D R, Babbitt P C, Gerlt J A, Rayment I
The Institute for Enzyme Research, Department of Biochemistry, University of Wisconsin, Madison 53706, USA.
Biochemistry. 1998 Oct 13;37(41):14358-68. doi: 10.1021/bi981123n.
The structure of (D)-glucarate dehydratase from Pseudomonas putida (GlucD) has been solved at 2.3 A resolution by multiple isomorphous replacement and refined to a final R-factor of 19.0%. The protein crystallizes in the space group I222 with one subunit in the asymmetric unit. The unit cell dimensions are a = 69.6 A, b = 108.8 A, and c = 122.6 A. The crystals were grown using the batch method where the primary precipitant was poly(ethylene glycol) 1000. The structure reveals that GlucD is a tetramer of four identical polypeptides, each containing 451 residues. The structure was determined without a bound substrate or substrate analogue. Three disordered regions are noted: the N-terminus through residue 11, a loop containing residues 99 through 110, and the C-terminus from residue 423. On the basis of primary sequence alignments, we previously concluded that GlucD is a member of the mandelate racemase (MR) subfamily of the enolase superfamily [Babbitt, P. C., Hasson, M. S., Wedekind, J. E., Palmer, D. R. J., Barrett, W. C., Reed, G. J., Rayment, I., Ringe, D., Kenyon, G. L., and Gerlt, J. A. (1996) Biochemistry 35, 16489-16501]. This prediction is now verified, since the overall fold of GlucD is strikingly similar to those of MR, muconate lactonizing enzyme I, and enolase. Also, many of the active site residues of GlucD can be superimposed on those found in the active site of MR. The implications of this structure on the evolution of catalysis in the enolase superfamily are discussed.
通过多同晶置换法，已解析出恶臭假单胞菌（Pseudomonas putida）的（D）-葡萄糖二酸脱水酶（GlucD）的结构，分辨率为2.3埃，并将其精修至最终R因子为19.0%。该蛋白质在空间群I222中结晶，不对称单元中有一个亚基。晶胞尺寸为a = 69.6埃，b = 108.8埃，c = 122.6埃。晶体采用分批法生长，主要沉淀剂为聚乙二醇1000。结构显示GlucD是由四个相同多肽组成的四聚体，每个多肽含有451个残基。该结构是在没有结合底物或底物类似物的情况下确定的。注意到三个无序区域：N端至第11位残基、包含第99至110位残基的环以及第423位残基开始的C端。基于一级序列比对，我们之前得出结论，GlucD是烯醇酶超家族中扁桃酸消旋酶（MR）亚家族的成员[巴比特，P.C.，哈森，M.S.，韦德金德，J.E.，帕尔默，D.R.J.，巴雷特，W.C.，里德，G.J.，雷门特，I.，林格，D.，凯尼恩，G.L.，和格尔特，J.A.（1996年）《生物化学》35，16489 - 16501]。现在这一预测得到了验证，因为GlucD的整体折叠与MR、粘康酸内酯化酶I和烯醇酶的折叠惊人地相似。此外，GlucD的许多活性位点残基可以与MR活性位点中的残基重叠。讨论了该结构对烯醇酶超家族催化进化的影响。
