Six David A, Carty Sherry M, Guan Ziqiang, Raetz Christian R H
Department of Biochemistry, Duke University Medical Center, P.O. Box 3711, Durham, North Carolina 27710, USA.
Biochemistry. 2008 Aug 19;47(33):8623-37. doi: 10.1021/bi800873n. Epub 2008 Jul 26.
Escherichia coli lipid A is a hexaacylated disaccharide of glucosamine with secondary laurate and myristate chains on the distal unit. Hexaacylated lipid A is a potent agonist of human Toll-like receptor 4, whereas its tetra- and pentaacylated precursors are antagonists. The inner membrane enzyme LpxL transfers laurate from lauroyl-acyl carrier protein to the 2'- R-3-hydroxymyristate moiety of the tetraacylated lipid A precursor Kdo 2-lipid IV A. LpxL has now been overexpressed, solubilized with n-dodecyl beta- d-maltopyranoside (DDM), and purified to homogeneity. LpxL migration on a gel filtration column is consistent with a molecular mass of 80 kDa, suggestive of an LpxL monomer (36 kDa) embedded in a DDM micelle. Mass spectrometry showed that deformylated LpxL was the predominant species, noncovalently bound to as many as 12 DDM molecules. Purified LpxL catalyzed not only the formation in vitro of Kdo 2-(lauroyl)-lipid IV A but also a slow second acylation, generating Kdo 2-(dilauroyl)-lipid IV A. Consistent with the Kdo dependence of crude LpxL in membranes, Kdo 2-lipid IV A is preferred 6000-fold over lipid IV A by the pure enzyme. Sequence comparisons suggest that LpxL shares distant homology with the glycerol-3-phosphate acyltransferase (GPAT) family, including a putative catalytic dyad located in a conserved H(X) 4D/E motif. Mutation of H132 or E137 to alanine reduces specific activity by over 3 orders of magnitude. Like many GPATs, LpxL can also utilize acyl-CoA as an alternative acyl donor, albeit at a slower rate. Our results show that the acyltransferases that generate the secondary acyl chains of lipid A are members of the GPAT family and set the stage for structural studies.
大肠杆菌脂多糖A是一种氨基葡萄糖的六酰化二糖，在远端单元上带有仲月桂酸酯和肉豆蔻酸酯链。六酰化脂多糖A是人类Toll样受体4的强效激动剂，而其四酰化和五酰化前体则是拮抗剂。内膜酶LpxL将月桂酸从月桂酰-酰基载体蛋白转移到四酰化脂多糖A前体Kdo 2-脂多糖IV A的2'-R-3-羟基肉豆蔻酸部分。LpxL现已过表达，用正十二烷基-β-D-麦芽糖苷（DDM）溶解，并纯化至同质。LpxL在凝胶过滤柱上的迁移与80 kDa的分子量一致，提示LpxL单体（36 kDa）嵌入DDM胶束中。质谱分析表明，去甲酰化的LpxL是主要形式，与多达12个DDM分子非共价结合。纯化的LpxL不仅催化体外形成Kdo 2-（月桂酰）-脂多糖IV A，还催化缓慢的第二次酰化反应，生成Kdo 2-（二月桂酰）-脂多糖IV A。与膜中粗LpxL对Kdo的依赖性一致，纯酶对Kdo 2-脂多糖IV A的偏好性比对脂多糖IV A高6000倍。序列比较表明，LpxL与甘油-3-磷酸酰基转移酶（GPAT）家族有远缘同源性，包括位于保守的H(X)4D/E基序中的一个假定催化二元组。将H132或E137突变为丙氨酸会使比活性降低超过3个数量级。与许多GPAT一样，LpxL也可以利用酰基辅酶A作为替代酰基供体，尽管速率较慢。我们的结果表明，产生脂多糖A二级酰基链的酰基转移酶是GPAT家族的成员，并为结构研究奠定了基础。
