Enzymatic synthesis of lipopolysaccharide in Escherichia coli -: Purification and properties of heptosyltransferase 1
Enzymatic synthesis of lipopolysaccharide in Escherichia coli -: Purification and properties of heptosyltransferase 1
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DOI：
10.1074/jbc.273.5.2799
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发表时间：
1998-01-30
影响因子：
4.8
通讯作者：
Raetz, CRH
中科院分区：
文献类型：
--
作者：
Kadrmas, JL;Raetz, CRH
由大肠杆菌的rfaC（waaC）基因编码的庚糖基转移酶I被认为将L-甘油-D-甘露-庚糖添加到脂多糖核心的内部3-脱氧-D-甘露-辛酮糖酸（Kdo）残基。从rfaC缺陷突变体分离的脂多糖缺乏庚糖和庚糖远端的所有其他糖。推定的供体ADP-L-甘油-D-甘露-庚糖从未被完全表征，并且不容易获得。在细胞提取物中，类似物ADP-甘露糖可以作为受体Kdo（2）-脂质IVA的RfaC催化糖基化的替代供体。使用过表达RfaC类似于15，000倍的T7启动子构建体，该酶已被纯化至接近同质。NH 2-末端测序证实纯化的酶是rfaC基因产物。亚基分子量为36 kDa，酶活性取决于Triton X-100的存在，在pH 7.5时最大。ADP-甘露糖的表观K-m（在第二种底物接近饱和浓度下测定）为1.5 mM，Kdo（2）-脂质IVA的表观K-m为4.5 μ M。在乙酸钠和1%十二烷基硫酸钠存在下，在100 ℃下RfaC反应产物的化学水解产生与Kdo（2）-脂质IVA的内部Kdo残基一致的片段作为甘露糖基化位点。类似物Kdo-脂质IVA作为受体发挥作用，但甘露糖基化速率小于Kdo（2）-脂质IVA的1%。纯化的酶与ADP-葡萄糖、GDP-甘露糖、UDP-葡萄糖或UDP-半乳糖不显示活性。由RfaC催化的Kdo（2）-脂质IVA的甘露糖基化以高产率进行，并且可用于脂多糖类似物的合成。纯RfaC也可与Kdo（2）-[4 ′ P-32]脂质IVA一起用于测定从野生型细胞分离的粗低分子量级分中的生理供体（推测为ADP-L-甘油-D-甘露-庚糖）。
Heptosyltransferase I, encoded by the rfaC(waaC) gene of Escherichia coli, is thought to add L-glycero-D-manno-heptose to the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide core. Lipopolysaccharide isolated from mutants defective in rfaC lack heptose and all other sugars distal to heptose. The putative donor, ADP-L-glycero-D-manno-heptose, has never been fully characterized and is not readily available, In cell extracts, the analog ADP-mannose can serve as an alternative donor for RfaC-catalyzed glycosylation of the acceptor, Kdo(2)-lipid IVA. Using a T7 promoter construct that overexpresses RfaC similar to 15,000-fold, the enzyme has been purified to near homogeneity. NH2-terminal sequencing confirms that the purified enzyme is the rfaC gene product. The subunit molecular mass is 36 kDa, Enzymatic activity is dependent upon the presence of Triton X-100 and is maximal at pH 7.5. The apparent K-m (determined at near saturating concentrations of the second substrate) is 1.5 mM for ADP-mannose and 4.5 mu M for Kdo(2)-lipid IVA. Chemical hydrolysis of the RfaC reaction product at 100 degrees C in the presence of sodium acetate and 1% sodium dodecyl sulfate generates fragments consistent with the inner Kdo residue of Kdo(2)-lipid IVA as the site of mannosylation, The analog, Kdo-lipid IVA, functions as an acceptor, but is mannosylated at less than 1% the rate of Kdo(2)-lipid IVA. The purified enzyme displays no activity with ADP-glucose, GDP-mannose, UDP-glucose, or UDP-galactose. Mannosylation of Kdo(2)-lipid IVA catalyzed by RfaC proceeds in high yield and may be useful for the synthesis of lipopolysaccharide analogs. Pure RfaC can also be used together with Kdo(2)-[4'P-32]lipid IVA to assay for the physiological donor (presumably ADP-L-glycero-D-manno-heptose) in a crude, low molecular weight fraction isolated from wild type cells.