The crystal structure of Bacillus cereus phosphonoacetaldehyde hydrolase:: Insight into catalysis of phosphorus bond cleavage and catalytic diversification within the HAD enzyme superfamily
The crystal structure of Bacillus cereus phosphonoacetaldehyde hydrolase:: Insight into catalysis of phosphorus bond cleavage and catalytic diversification within the HAD enzyme superfamily
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DOI：
10.1021/bi001171j
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发表时间：
2000-08-29
期刊：
影响因子：
2.9
通讯作者：
Allen, KN
中科院分区：
文献类型：
--
作者：
Morais, MC;Zhang, WH;Allen, KN
膦酰乙醛水解酶（磷酸酶）使用Mg（II）作为辅因子催化膦酰乙醛水解成乙醛和磷酸盐。该反应通过一种新的二价催化机制进行，其中活性位点亲核试剂从Lys 53和膦酰基乙醛形成的席夫碱中间体中提取磷酰基。在这项研究中，X-射线晶体结构的蜡状芽孢杆菌磷酸酶同型二聚体与磷酸盐（产品）类似物钨酸盐（Ki = 50 μ M）和镁（II）辅因子的复合确定为3.0埃分辨率与R-cryst = 0.248和R-free = 0.284。每个单体由α/β核心结构域组成，所述α/β核心结构域由被六个α-螺旋包围的位于中心的六链平行β-折叠组成。两个柔性的溶剂化接头连接到由反平行的五螺旋束组成的小帽结构域（残基21-99）。亚基-亚基界面由来自各自核心结构域的两个α 8螺旋的对称包装形成，通过源自成对Met 171、Trp 164、Tyr 162、Tyr 167和Tyr 176侧链的去溶剂化的疏水效应而稳定。活性位点位于每个亚基的结构域-结构域界面。形成Lys 53的席夫碱位于帽域上，而钨酸盐和Mg（II）结合到核心域。Mg（II）配体包括钨酸盐配体的两个氧，Asp 12和Asp 186的羧酸盐的一个氧。Ala 14的骨架羰基氧，以及与Asp 190和Thr 187的羧酸根形成氢键的水。Arg 160的胍基结合钨酸盐和建议的亲核试剂Asp 12，Asp 12适合于在钨原子上进行在线攻击。核心结构域残基Tyr 128和帽结构域残基Cys 22和Lys 53的侧链位于附近。Asp 12作为活性位点亲核试剂的身份通过观察到Asp 12 Ala取代导致的催化活性的去除进一步证明。在磷酸酶和2-卤酸脱卤酶的HAD酶超家族中观察到的骨架折叠的相似性表明共同的祖先。这两种结构的叠加揭示了一个保守的活性位点支架具有不同的催化站。HAD超家族的脱卤酶、磷酸酶、磷酸酶和磷酸变位酶在这些位点上使用极性氨基酸残基的分析表明，古老酶祖先的活性位点可能已经多样化，用于催化C-X、P-C和P-O键断裂反应。
Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and phosphate using Mg(II) as cofactor. The reaction proceeds via a novel bicovalent catalytic mechanism in which an active-site nucleophile abstracts the phosphoryl group from the Schiff-base intermediate formed from Lys53 and phosphonoacetaldehyde. In this study, the X-ray crystal structure of the Bacillus cereus phosphonatase homodimer complexed with the phosphate (product) analogue tungstate (K-i = 50 mu M) and the Mg(II) cofactor was determined to 3.0 Angstrom resolution with an R-cryst = 0.248 and R-free = 0.284. Each monomer is made up of an alpha/beta core domain consisting of a centrally located six-stranded parallel beta-sheet surrounded by six alpha-helices. Two flexible, solvated linkers connect to a small cap domain (residues 21-99) that consists of an antiparallel, five-helix bundle. The subunit-subunit interface, formed by the symmetrical packing of the two alpha 8 helices from the respective core domains, is stabilized through the hydrophobic effect derived from the desolvation of paired Met171, Trp 164, Tyr162, Tyr167, and Tyr176 side chains. The active site is located at the domain-domain interface of each subunit. The Schiff base forming Lys53 is positioned on the cap domain while tungstate and Mg(II) are bound to the core domain. Mg(II) ligands include two oxygens of the tungstate ligand, one oxygen of the carboxylates of Asp12 and Asp186. the backbone carbonyl oxygen of Ala14, and a water that forms a hydrogen bond with the carboxylate of Asp190 and Thr187. The guanidinium group of Arg160 binds tungstate and the proposed nucleophile Asp12, which is suitably positioned for in-line attack at the tungsten atom. The side chains of the core domain residue Tyr128 and the cap domain residues Cys22 and Lys53 are located nearby. The identity of Asp12 as the active-site nucleophile was further evidenced by the observed removal of catalytic activity resulting from Asp12Ala substitution. The similarity of backbone folds observed in phosphonatase and the 2-haloacid dehalogenase of the HAD enzyme superfamily indicated common ancestry. Superposition of the two structures revealed a conserved active-site scaffold having distinct catalytic stations. Analysis of the usage of polar amino acid residues at these stations by the dehalogenases, phosphonatases, phosphatases, and phosphomutases of the HAD superfamily suggests possible ways in which the active site of an ancient enzyme ancestor might have been diversified for catalysis of C-X, P-C, and P-O bond cleavage reactions.