Dotson G D, Kaltashov I A, Cotter R J, Raetz C R
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Bacteriol. 1998 Jan;180(2):330-7. doi: 10.1128/JB.180.2.330-337.1998.
UDP-N-acetylglucosamine-3-O-acyltransferase (UDP-GlcNAc acyltransferase) catalyzes the first step of lipid A biosynthesis (M. S. Anderson and C. R. H. Raetz, J. Biol. Chem. 262:5159-5169, 1987). We here report the isolation of the lpxA gene of Pseudomonas aeruginosa from a library of Pseudomonas strain PAO1 expressed in Escherichia coli LE392 (J. Lightfoot and J. S. Lam, J. Bacteriol. 173:5624-5630, 1991). Pseudomonas lpxA encodes a 10-carbon-specific UDP-GlcNAc acyltransferase, whereas the E. coli transferase is selective for a 14-carbon acyl chain. Recombinant cosmid 1137 enabled production of a 3-hydroxydecanoyl-specific UDP-GlcNAc acyltransferase in E. coli. It was identified by assaying lysozyme-EDTA lysates of individual members of the library with 3-hydroxydecanoyl-acyl carrier protein (ACP) as the substrate. Cosmid 1137 contained a 20-kb insert of P. aeruginosa DNA. The lpxA gene region was localized to a 1.3-kb SalI-PstI fragment. Sequencing revealed that it contains one complete open reading frame (777 bp) encoding a new lpxA homolog. The predicted Pseudomonas LpxA is 258 amino acids long and contains 21 complete hexapeptide repeating units, spaced in approximately the same manner as the 24 repeats of E. coli LpxA. The P. aeruginosa UDP-GlcNAc acyltransferase is 54% identical and 67% similar to the E. coli enzyme. A plasmid (pGD3) containing the 1.3-kb SalI-PstI fragment complemented E. coli RO138, a temperature-sensitive mutant harboring lpxA2. LpxA assays of extracts of this construct indicated that it is > 1,000-fold more selective for 3-hydroxydecanoyl-ACP than for 3-hydroxymyristoyl-ACP. Mass spectrometry of lipid A isolated from this strain by hydrolysis at pH 4.5 revealed [M-H]- 1,684.5 (versus 1,796.5 for wild-type lipid A), consistent with 3-hydroxydecanoate rather than 3-hydroxymyristate at positions 3 and 3'.
UDP-N-乙酰葡糖胺-3-O-酰基转移酶（UDP-GlcNAc酰基转移酶）催化脂多糖A生物合成的第一步（M. S. 安德森和C. R. H. 雷茨，《生物化学杂志》262:5159 - 5169，1987年）。我们在此报告从在大肠杆菌LE392中表达的铜绿假单胞菌菌株PAO1文库中分离出铜绿假单胞菌的lpxA基因（J. 莱特富特和J. S. 林，《细菌学杂志》173:5624 - 5630，1991年）。铜绿假单胞菌lpxA编码一种10碳特异性UDP-GlcNAc酰基转移酶，而大肠杆菌转移酶对14碳酰基链具有选择性。重组黏粒1137使大肠杆菌能够产生3-羟基癸酰特异性UDP-GlcNAc酰基转移酶。通过以3-羟基癸酰-酰基载体蛋白（ACP）为底物检测文库中各个成员的溶菌酶-EDTA裂解物来鉴定它。黏粒1137包含一个20 kb的铜绿假单胞菌DNA插入片段。lpxA基因区域定位于一个1.3 kb的SalI - PstI片段。测序显示它包含一个完整的开放阅读框（777 bp），编码一个新的lpxA同源物。预测的铜绿假单胞菌LpxA长258个氨基酸，包含21个完整的六肽重复单元，其间隔方式与大肠杆菌LpxA的24个重复单元大致相同。铜绿假单胞菌UDP-GlcNAc酰基转移酶与大肠杆菌酶的同一性为54%，相似性为67%。含有1.3 kb SalI - PstI片段的质粒（pGD3）互补了大肠杆菌RO138，它是一个携带lpxA2的温度敏感突变体。对该构建体提取物的LpxA测定表明，它对3-羟基癸酰-ACP的选择性比对3-羟基肉豆蔻酰-ACP高1000倍以上。通过在pH 4.5下水解从该菌株分离的脂多糖A的质谱分析显示[M - H]-为1684.5（野生型脂多糖A为1796.5），这与3和3'位上是3-羟基癸酸而不是3-羟基肉豆蔻酸一致。
