Silvaggi Nicholas R, Zhang Chunchun, Lu Zhibing, Dai Jianying, Dunaway-Mariano Debra, Allen Karen N
Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA.
J Biol Chem. 2006 May 26;281(21):14918-26. doi: 10.1074/jbc.M601505200. Epub 2006 Mar 15.
Congenital disorder of glycosylation type 1a (CDG-1a) is a congenital disease characterized by severe defects in nervous system development. It is caused by mutations in alpha-phosphomannomutase (of which there are two isozymes, alpha-PMM1 and alpha-PPM2). Here we report the x-ray crystal structures of human alpha-PMM1 in the open conformation, with and without the bound substrate, alpha-D-mannose 1-phosphate. Alpha-PMM1, like most haloalkanoic acid dehalogenase superfamily (HADSF) members, consists of two domains, the cap and core, which open to bind substrate and then close to provide a solvent-exclusive environment for catalysis. The substrate phosphate group is observed at a positively charged site of the cap domain, rather than at the core domain phosphoryl-transfer site defined by the Asp(19) nucleophile and Mg(2+) cofactor. This suggests that substrate binds first to the cap and then is swept into the active site upon cap closure. The orientation of the acid/base residue Asp(21) suggests that alpha-phosphomannomutase (alpha-PMM) uses a different method of protecting the aspartylphosphate from hydrolysis than the HADSF member beta-phosphoglucomutase. It is hypothesized that the electrostatic repulsion of positive charges at the interface of the cap and core domains stabilizes alpha-PMM1 in the open conformation and that the negatively charged substrate binds to the cap, thereby facilitating its closure over the core domain. The two isozymes, alpha-PMM1 and alpha-PMM2, are shown to have a conserved active-site structure and to display similar kinetic properties. Analysis of the known mutation sites in the context of the structures reveals the genotype-phenotype relationship underlying CDG-1a.
1a型先天性糖基化障碍（CDG-1a）是一种先天性疾病，其特征是神经系统发育存在严重缺陷。它由α-磷酸甘露糖变位酶（有两种同工酶，α-PMM1和α-PPM2）的突变引起。在此，我们报告了人α-PMM1处于开放构象时的X射线晶体结构，包括结合和未结合底物α-D-甘露糖1-磷酸的情况。α-PMM1与大多数卤代烷酸脱卤酶超家族（HADSF）成员一样，由两个结构域组成，即帽状结构域和核心结构域，它们打开以结合底物，然后关闭以提供一个与溶剂隔绝的催化环境。底物磷酸基团出现在帽状结构域的一个带正电荷的位点，而不是由天冬氨酸（19）亲核试剂和镁（2+）辅因子定义的核心结构域磷酸转移位点。这表明底物首先结合到帽状结构域，然后在帽状结构域关闭时被扫入活性位点。酸碱残基天冬氨酸（21）的取向表明，α-磷酸甘露糖变位酶（α-PMM）与HADSF成员β-磷酸葡萄糖变位酶相比，采用了不同的方法来保护天冬氨酰磷酸不被水解。据推测，帽状结构域和核心结构域界面处正电荷的静电排斥使α-PMM1处于开放构象稳定，而带负电荷的底物与帽状结构域结合，从而促进其在核心结构域上关闭。两种同工酶α-PMM1和α-PMM2具有保守的活性位点结构，并表现出相似的动力学性质。在结构背景下对已知突变位点的分析揭示了CDG-1a潜在的基因型-表型关系。
