Breazeale Steven D, Ribeiro Anthony A, Raetz Christian R H
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 2003 Jul 4;278(27):24731-9. doi: 10.1074/jbc.M304043200. Epub 2003 Apr 18.
In Escherichia coli and Salmonella typhimurium, addition of the 4-amino-4-deoxy-l-arabinose (l-Ara4N) moiety to the phosphate group(s) of lipid A is required for resistance to polymyxin and cationic antimicrobial peptides. We have proposed previously (Breazeale, S. D., Ribeiro, A. A., and Raetz, C. R. H. (2002) J. Biol. Chem. 277, 2886-2896) a pathway for l-Ara4N biosynthesis that begins with the ArnA-catalyzed C-4" oxidation and C-6" decarboxylation of UDP-glucuronic acid, followed by the C-4" transamination of the product to generate the novel sugar nucleotide UDP-l-Ara4N. We now show that ArnB (PmrH) encodes the relevant aminotransferase. ArnB was overexpressed using a T7lac promoter-driven construct and shown to catalyze the reversible transfer of the amino group from glutamate to the acceptor, uridine 5'-(beta-l-threo-pentapyranosyl-4"-ulose diphosphate), the intermediate that is synthesized by ArnA from UDP-glucuronic acid. A 1.7-mg sample of the putative UDP-l-Ara4N product generated in vitro was purified by ion exchange chromatography, and its structure was confirmed by 1H and 13C NMR spectroscopy. ArnB, which is a cytoplasmic protein, was purified to homogeneity from an overproducing strain of E. coli and shown to contain a pyridoxal phosphate cofactor, as judged by ultraviolet/visible spectrophotometry. The pyridoxal phosphate was converted to the pyridoxamine form in the presence of excess glutamate. A simple quantitative radiochemical assay was developed for ArnB, which can be used to assay the enzyme either in the forward or the reverse direction. The enzyme is highly selective for glutamate as the amine donor, but the equilibrium constant in the direction of UDP-l-Ara4N formation is unfavorable (approximately 0.1). ArnB is a member of a very large family of aminotransferases, but closely related ArnB orthologs are present only in those bacteria capable of synthesizing lipid A species modified with the l-Ara4N moiety.
在大肠杆菌和鼠伤寒沙门氏菌中，脂质A的磷酸基团上添加4-氨基-4-脱氧-L-阿拉伯糖（L-Ara4N）部分是对多粘菌素和阳离子抗菌肽产生抗性所必需的。我们之前曾提出（Breazeale, S. D., Ribeiro, A. A., and Raetz, C. R. H. (2002) J. Biol. Chem. 277, 2886 - 2896）一条L-Ara4N生物合成途径，该途径始于ArnA催化的UDP-葡萄糖醛酸的C-4”氧化和C-6”脱羧，随后产物进行C-4”转氨作用以生成新型糖核苷酸UDP-L-Ara4N。我们现在表明ArnB（PmrH）编码相关的转氨酶。使用T7lac启动子驱动的构建体对ArnB进行过表达，并证明其催化氨基从谷氨酸可逆转移至受体尿苷5'-（β-L-苏式-戊吡喃糖基-4”-酮二磷酸），这是ArnA由UDP-葡萄糖醛酸合成的中间体。通过离子交换色谱法纯化了体外产生的1.7毫克推定的UDP-L-Ara4N产物样本，并通过1H和13C NMR光谱确认了其结构。ArnB是一种细胞质蛋白，从过量表达的大肠杆菌菌株中纯化至同质，并通过紫外/可见分光光度法判断其含有磷酸吡哆醛辅因子。在过量谷氨酸存在下，磷酸吡哆醛转化为吡哆胺形式。开发了一种针对ArnB的简单定量放射化学测定法，可用于正向或反向测定该酶。该酶对作为胺供体的谷氨酸具有高度选择性，但UDP-L-Ara4N形成方向的平衡常数不利（约为0.1）。ArnB是一个非常大的转氨酶家族的成员，但密切相关的ArnB直系同源物仅存在于那些能够合成用L-Ara4N部分修饰的脂质A种类的细菌中。
