Skinner M M, Puvathingal J M, Walter R L, Friedman A M
Department of Biological Sciences, Purdue University , West Lafayette, IN 47907, USA.
Structure. 2000 Nov 15;8(11):1189-201. doi: 10.1016/s0969-2126(00)00522-0.
Formation of isoaspartyl residues is one of several processes that damage proteins as they age. Protein L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) is a conserved and nearly ubiquitous enzyme that catalyzes the repair of proteins damaged by isoaspartyl formation.
We have determined the first structure of a PIMT from crystals of the T. maritima enzyme complexed to S-adenosyl-L-homocysteine (AdoHcy) and refined it to 1.8 A resolution. Although PIMT forms one structural unit, the protein can be divided functionally into three subdomains. The central subdomain closely resembles other S-adenosyl-L-methionine-dependent methyltransferases but bears a striking alteration of topological connectivity, which is not shared by any other member of this family. Rather than arranged as a mixed beta sheet with topology 6 upward arrow7 downward arrow5 upward arrow4 upward arrow1 upward arrow2 upward arrow3 upward arrow, the central sheet of PIMT is reorganized to 7 upward arrow6 downward arrow5 upward arrow4 upward arrow1 upward arrow2 upward arrow3 upward arrow. AdoHcy is largely buried between the N-terminal and central subdomains by a conserved and largely hydrophobic loop on one rim of the binding cleft, and a conserved Ser/Thr-rich beta strand on the other. The Ser/Thr-rich strand may provide hydrogen bonds for specific interactions with isoaspartyl substrates. The side chain of Ile-206, a conserved residue, crosses the cleft, restricting access to the donor methyl group to a deep well, the putative isoaspartyl methyl acceptor site.
The structure of PIMT reveals a unique modification of the methyltransferase fold along with a site for specific recognition of isoaspartyl substrates. The sequence conservation among PIMTs suggests that the current structure should prove a reliable model for understanding the repair of isoaspartyl damage in all organisms.
异天冬氨酸残基的形成是蛋白质老化过程中导致其损伤的几种机制之一。蛋白质L-异天冬氨酸（D-天冬氨酸）O-甲基转移酶（PIMT）是一种保守且几乎普遍存在的酶，它催化修复因异天冬氨酸形成而受损的蛋白质。
我们通过嗜热栖热菌（T. maritima）酶与S-腺苷-L-高半胱氨酸（AdoHcy）形成的复合物晶体，首次确定了PIMT的结构，并将其精修至1.8 Å分辨率。尽管PIMT形成一个结构单元，但该蛋白质在功能上可分为三个亚结构域。中央亚结构域与其他依赖S-腺苷-L-甲硫氨酸的甲基转移酶非常相似，但拓扑连接性有显著改变，该家族的其他成员均不具备这一特征。PIMT的中央片层并非以拓扑结构为6上箭头7下箭头5上箭头4上箭头1上箭头2上箭头3上箭头的混合β折叠形式排列，而是重新组织为7上箭头6下箭头5上箭头4上箭头1上箭头2上箭头3上箭头。AdoHcy主要通过结合裂隙一侧边缘上保守且大多为疏水的环以及另一侧富含丝氨酸/苏氨酸的β链，掩埋于N端和中央亚结构域之间。富含丝氨酸/苏氨酸的链可能为与异天冬氨酸底物的特异性相互作用提供氢键。保守残基异亮氨酸-206的侧链穿过裂隙，将供体甲基的 access限制在一个深阱中，即假定的异天冬氨酸甲基受体位点。
PIMT的结构揭示了甲基转移酶折叠的独特修饰以及异天冬氨酸底物特异性识别位点。PIMT之间的序列保守性表明，当前结构应能成为理解所有生物体中异天冬氨酸损伤修复的可靠模型。
