Garrett T A, Kadrmas J L, Raetz C R
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1997 Aug 29;272(35):21855-64. doi: 10.1074/jbc.272.35.21855.
The genes for seven of nine enzymes needed for the biosynthesis of Kdo2-lipid A (Re endotoxin) in Escherichia coli have been reported. We have now identified a novel gene encoding the lipid A 4'-kinase (the sixth step of the pathway). The 4'-kinase transfers the gamma-phosphate of ATP to the 4'-position of a tetraacyldisaccharide 1-phosphate intermediate (termed DS-1-P) to form tetraacyldisaccharide 1,4'-bis-phosphate (lipid IVA). The 4'-phosphate is required for the action of distal enzymes, such as Kdo transferase and also renders lipid A substructures active as endotoxin antagonists or mimetics. Lysates of E. coli generated using individual lambda clones from the ordered Kohara library were assayed for overproduction of 4'-kinase. Only one clone, [218]E1D1, which directed 2-2.5-fold overproduction, was identified. This construct contains 20 kilobase pairs of E. coli DNA from the vicinity of minute 21. Two genes related to the lipid A system map in this region: msbA, encoding a putative translocator, and kdsB, the structural gene for CMP-Kdo synthase. msbA forms an operon with a downstream, essential open reading frame of unknown function, designated orfE. orfE was cloned into a T7 expression system. Washed membranes from cells overexpressing orfE display approximately 2000-fold higher specific activity of 4'-kinase than membranes from cells with vector alone. Membranes containing recombinant, overexpressed 4'-kinase (but not membranes with wild-type kinase levels) efficiently phosphorylate three DS-1-P analogs: 3-aza-DS-1-P, base-treated DS-1-P, and base-treated 3-aza-DS-1-P. A synthetic hexaacylated DS-1-P analog, compound 505, can also be phosphorylated by membranes from the overproducer, yielding [4'-32P] lipid A (endotoxin). The overexpressed lipid A 4'-kinase is very useful for making new 4'-phosphorylated lipid A analogs with potential utility as endotoxin mimetics or antagonists. We suggest that orfE is the structural gene for the 4'-kinase and that it be redesignated lpxK.
大肠杆菌中Kdo2-脂质A（脂多糖内毒素）生物合成所需的9种酶中的7种基因已被报道。我们现已鉴定出一个编码脂质A 4'-激酶（该途径的第六步）的新基因。4'-激酶将ATP的γ-磷酸基团转移到四酰基二糖1-磷酸中间体（称为DS-1-P）的4'-位，形成四酰基二糖1,4'-双磷酸（脂质IVA）。4'-磷酸对于远端酶（如Kdo转移酶）的作用是必需的，并且还使脂质A亚结构作为内毒素拮抗剂或模拟物具有活性。使用来自有序小原文库的单个λ克隆产生的大肠杆菌裂解物用于检测4'-激酶的过量表达。仅鉴定出一个克隆[218]E1D1，其指导2 - 2.5倍的过量表达。该构建体包含来自21分钟附近的20千碱基对的大肠杆菌DNA。在该区域定位有两个与脂质A系统相关的基因：msbA，编码一个假定的转运体；以及kdsB，CMP-Kdo合酶的结构基因。msbA与一个下游的、功能未知的必需开放阅读框形成一个操纵子，命名为orfE。orfE被克隆到一个T7表达系统中。过表达orfE的细胞洗涤后的膜显示出比仅含载体的细胞的膜高约2000倍的4'-激酶比活性。含有重组的、过表达的4'-激酶的膜（但不是具有野生型激酶水平的膜）能有效地磷酸化三种DS-1-P类似物：3-氮杂-DS-1-P、碱处理的DS-1-P和碱处理的3-氮杂-DS-1-P。一种合成的六酰化DS-1-P类似物化合物505也可被过量表达菌的膜磷酸化，产生[4'-32P]脂质A（内毒素）。过表达的脂质A 4'-激酶对于制备具有作为内毒素模拟物或拮抗剂潜在用途的新的4'-磷酸化脂质A类似物非常有用。我们认为orfE是4'-激酶的结构基因，并建议将其重新命名为lpxK。
