The UDP-diacylglucosamine Pyrophosphohydrolase LpxH in Lipid A Biosynthesis Utilizes Mn2+ Cluster for Catalysis
The UDP-diacylglucosamine Pyrophosphohydrolase LpxH in Lipid A Biosynthesis Utilizes Mn2+ Cluster for Catalysis
复制标题
DOI：
10.1074/jbc.m113.497636
复制
发表时间：
2013-09-20
影响因子：
4.8
通讯作者：
Zhou, Pei
中科院分区：
文献类型：
--
作者：
Young, Hayley E.;Donohue, Matthew P.;Zhou, Pei
在大肠杆菌和大多数β-和γ-蛋白细菌中，脂A生物合成的第四步，即UDP-2，3-二酰基-GlcN焦磷酸基团的裂解是由LpxH进行的。LpxH先前被认为包含在钙调神经磷酸酯酶(CLP)金属酶家族中发现的特征基序；然而，它裂解焦磷酸键而不是磷酸酯键，其底物包含更常见的Nudex家族而不是CLP家族的核苷二磷酸部分。此外，生化数据的范围未能在酶分析中显示出显著的金属活性水平，这与金属酶的行为不一致。在这里，我们报道了流感嗜血杆菌LpxH(HiLpxH)的克隆、纯化和详细的酶学性质。在Mn2+存在下，HiLpxH对水解酶活性有600多倍的刺激作用。EPR研究表明，LpxH中存在一个Mn2+团簇。最后，CLP家族保守的金属结合基序中残基的点突变极大地抑制了HiLpxH的活性，突显了它们在酶功能中的重要性。与前人对LpxH的分析相反，我们发现HiLpxH不服从表面稀释动力学。总体而言，我们的工作明确地确立了LpxH是一种钙调神经磷酸酶，含有一个由保守残基配位的Mn2+簇。这些结果为进一步研究该酶的结构和设计针对脂质A生物合成的新型抗生素奠定了基础。
In Escherichia coli and the majority of beta- and gamma-proteobacteria, the fourth step of lipid A biosynthesis, i.e. cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN, is carried out by LpxH. LpxH has been previously suggested to contain signature motifs found in the calcineurin-like phosphoesterase (CLP) family of metalloenzymes; however, it cleaves a pyrophosphate bond instead of a phosphoester bond, and its substrate contains nucleoside diphosphate moieties more common to the Nudix family rather than to the CLP family. Furthermore, the extent of biochemical data fails to demonstrate a significant level of metal activation in enzymatic assays, which is inconsistent with the behavior of a metalloenzyme. Here, we report cloning, purification, and detailed enzymatic characterization of Haemophilus influenzae LpxH (HiLpxH). HiLpxH shows over 600-fold stimulation of hydrolase activity in the presence of Mn2+. EPR studies reveal the presence of a Mn2+ cluster in LpxH. Finally, point mutants of residues in the conserved metal-binding motifs of the CLP family greatly inhibit HiLpxH activity, highlighting their importance in enzyme function. Contrary to previous analyses of LpxH, we find HiLpxH does not obey surface dilution kinetics. Overall, our work unambiguously establishes LpxH as a calcineurin-like phosphoesterase containing a Mn2+ cluster coordinated by conserved residues. These results set the scene for further structural investigation of the enzyme and for design of novel antibiotics targeting lipid A biosynthesis.