Brozek K A, Carlson R W, Raetz C R
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1996 Dec 13;271(50):32126-36. doi: 10.1074/jbc.271.50.32126.
Lipid A, the hydrophobic anchor of lipopolysaccharides in the outer membranes of Gram-negative bacteria, varies in structure among different Rhizobiaceae. The Rhizobium meliloti lipid A backbone, like that of Escherichia coli, is a beta1'-6-linked glucosamine disaccharide that is phosphorylated at positions 1 and 4'. Rhizobium leguminosarum lipid A lacks both phosphates, but contains aminogluconate in place of the proximal glucosamine 1-phosphate, and galacturonic acid instead of the 4'-phosphate. A peculiar feature of the lipid As of all Rhizobiaceae is acylation with 27-hydroxyoctacosanoic acid, a long hydroxylated fatty acid not found in E. coli. We now describe an in vitro system, consisting of a membrane enzyme and a cytosolic acyl donor from R. leguminosarum, that transfers 27-hydroxyoctacosanoic acid to (Kdo)2-lipid IVA, a key lipid A precursor common to both E. coli and R. leguminosarum. The 27-hydroxyoctacosanoic acid moiety was detected in the lipid product by mass spectrometry. The membrane enzyme required the presence of Kdo residues in the acceptor substrate for activity. The cytosolic acyl donor was purified from wild-type R. leguminosarum using the acylation of (Kdo)2-[4'-32P]-lipid IVA as the assay. Amino-terminal sequencing of the purified acyl donor revealed an exact 19-amino acid match with a partially sequenced gene (orf*) of R. leguminosarum. Orf* contains the consensus sequence, DSLD, for attachment of 4'-phosphopantetheine. When the entire orf* gene was sequenced, it was found to encode a protein of 92 amino acids. Orf* is a new kind of acyl carrier protein because it is only approximately 25% identical both to the constitutive acyl carrier protein (AcpP) and to the inducible acyl carrier protein (NodF) of R. leguminosarum. Mass spectrometry of purified active Orf* confirmed the presence of 4'-phosphopantetheine and 27-hydroxyoctacosanoic acid in the major species. Smaller mass peaks indicative of Orf* acylation with hydroxylated 20, 22, 24, and 26 carbon fatty acids were also observed. Given the specialized function of Orf* in lipid A acylation, we suggest the new designation AcpXL.
脂多糖A是革兰氏阴性菌外膜中脂多糖的疏水锚定物，在不同的根瘤菌科细菌中结构各异。苜蓿根瘤菌的脂多糖A骨架与大肠杆菌的一样，是一种β1'-6连接的葡糖胺二糖，在1位和4'位磷酸化。豌豆根瘤菌的脂多糖A既没有磷酸基团，近端的葡糖胺1-磷酸被氨基葡糖酸取代，4'-磷酸被半乳糖醛酸取代。所有根瘤菌科细菌的脂多糖A的一个独特特征是被27-羟基二十八烷酸酰化，这是一种在大肠杆菌中未发现的长链羟基脂肪酸。我们现在描述一个体外系统，它由豌豆根瘤菌的一种膜酶和一种胞质酰基供体组成，该系统能将27-羟基二十八烷酸转移到（Kdo）2-脂IV A上，（Kdo）2-脂IV A是大肠杆菌和豌豆根瘤菌共有的一种关键脂多糖A前体。通过质谱在脂质产物中检测到了27-羟基二十八烷酸部分。膜酶的活性需要受体底物中存在Kdo残基。胞质酰基供体是从野生型豌豆根瘤菌中纯化得到的，以（Kdo）2-[4'-32P]-脂IV A的酰化反应作为检测方法。对纯化的酰基供体进行氨基末端测序，发现与豌豆根瘤菌一个部分测序的基因（orf*）有完全匹配的19个氨基酸序列。Orf包含用于连接4'-磷酸泛酰巯基乙胺的共有序列DSLD。对整个orf基因进行测序时，发现它编码一个92个氨基酸的蛋白质。Orf是一种新型的酰基载体蛋白，因为它与豌豆根瘤菌的组成型酰基载体蛋白（AcpP）和诱导型酰基载体蛋白（NodF）的相似度仅约为25%。对纯化的活性Orf进行质谱分析，证实主要物种中存在4'-磷酸泛酰巯基乙胺和27-羟基二十八烷酸。还观察到较小的质谱峰，表明Orf被羟基化的20、22、24和26碳脂肪酸酰化。鉴于Orf在脂多糖A酰化中的特殊功能，我们建议将其重新命名为AcpXL。
