Lahiri Sushmita D, Zhang Guofeng, Dunaway-Mariano Debra, Allen Karen N
Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118-2394, USA.
Bioorg Chem. 2006 Dec;34(6):394-409. doi: 10.1016/j.bioorg.2006.09.007. Epub 2006 Oct 27.
Phosphonatase functions in the 2-aminoethylphosphonate (AEP) degradation pathway of bacteria, catalyzing the hydrolysis of the C-P bond in phosphonoacetaldehyde (Pald) via formation of a bi-covalent Lys53ethylenamine/Asp12 aspartylphosphate intermediate. Because phosphonatase is a member of the haloacid dehalogenase superfamily, a family predominantly comprised of phosphatases, the question arises as to how this new catalytic activity evolved. The source of general acid-base catalysis for Schiff-base formation and aspartylphosphate hydrolysis was probed using pH-rate profile analysis of active-site mutants and X-ray crystallographic analysis of modified forms of the enzyme. The 2.9 A X-ray crystal structure of the mutant Lys53Arg complexed with Mg2+ and phosphate shows that the equilibrium between the open and the closed conformation is disrupted, favoring the open conformation. Thus, proton dissociation from the cap domain Lys53 is required for cap domain-core domain closure. The likely recipient of the Lys53 proton is a water-His56 pair that serves to relay the proton to the carbonyl oxygen of the phosphonoacetaldehyde (Pald) substrate upon addition of the Lys53. The pH-rate profile analysis of active-site mutants was carried out to test this proposal. The proximal core domain residues Cys22 and Tyr128 were ruled out, and the role of cap domain His56 was supported by the results. The X-ray crystallographic structure of wild-type phosphonatase reduced with NaBH4 in the presence of Pald was determined at 2.4A resolution to reveal N epsilon-ethyl-Lys53 juxtaposed with a sulfate ligand bound in the phosphate site. The position of the C2 of the N-ethyl group in this structure is consistent with the hypothesis that the cap domain N epsilon-ethylenamine-Lys53 functions as a general base in the hydrolysis of the aspartylphosphate bi-covalent enzyme intermediate. Because the enzyme residues proposed to play a key role in P-C bond cleavage are localized on the cap domain, this domain appears to have evolved to support the diversification of the HAD phosphatase core domain for catalysis of hydrolytic P-C bond cleavage.
磷酸酶在细菌的2-氨基乙基膦酸（AEP）降解途径中发挥作用，通过形成双共价Lys53乙撑胺/ Asp12天冬氨酰磷酸中间体催化膦酰乙醛（Pald）中C-P键的水解。由于磷酸酶是卤代酸脱卤酶超家族的成员，该家族主要由磷酸酶组成，因此出现了这种新催化活性如何进化的问题。使用活性位点突变体的pH-速率曲线分析和酶修饰形式的X射线晶体学分析来探究席夫碱形成和天冬氨酰磷酸水解的酸碱催化来源。与Mg2+和磷酸盐复合的突变体Lys53Arg的2.9 Å X射线晶体结构表明，开放构象和封闭构象之间的平衡被破坏，有利于开放构象。因此，帽结构域Lys53的质子解离是帽结构域-核心结构域闭合所必需的。Lys53质子的可能受体是水-His56对，在添加Lys53后，该对用于将质子传递到膦酰乙醛（Pald）底物的羰基氧上。对活性位点突变体进行了pH-速率曲线分析以验证该提议。排除了近端核心结构域残基Cys22和Tyr128，结果支持了帽结构域His56的作用。在Pald存在下用NaBH4还原的野生型磷酸酶的X射线晶体结构在2.4 Å分辨率下确定，以揭示Nε-乙基-Lys53与结合在磷酸盐位点的硫酸盐配体并列。该结构中N-乙基的C2位置与帽结构域Nε-乙撑胺-Lys53在天冬氨酰磷酸双共价酶中间体水解中作为通用碱起作用的假设一致。由于提议在P-C键裂解中起关键作用的酶残基位于帽结构域上，该结构域似乎已经进化以支持HAD磷酸酶核心结构域在催化水解P-C键裂解方面的多样化。
