Oxidative decarboxylation of UDP-glucuronic acid in extracts of polymyxin-resistant Escherichia coli -: Origin of lipid a species modified with 4-amino-4-deoxy-L-arabinose
Oxidative decarboxylation of UDP-glucuronic acid in extracts of polymyxin-resistant Escherichia coli -: Origin of lipid a species modified with 4-amino-4-deoxy-L-arabinose
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DOI：
10.1074/jbc.m109377200
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发表时间：
2002-01-25
影响因子：
4.8
通讯作者：
Raetz, CRH
中科院分区：
文献类型：
--
作者：
Breazeale, SD;Ribeiro, AA;Raetz, CRH
将4-氨基-4-脱氧-L-阿拉伯糖（L-Ara 4 N）部分添加至脂质A的磷酸基团涉及细菌对先天免疫系统的多粘菌素和阳离子抗微生物肽的抗性。鼠伤寒沙门氏菌pmrE和pmrF基因座产物的序列，这两者都是多粘菌素抗性所需的，最近使我们提出了从UDP-葡糖醛酸生物合成L-Ara 4 N的途径（Zhou，Z.，林，S.，科特河J.，和Raetz，C. R. H.（1999）J.Biol.Chem.274，18503-18514）。我们现在报道，当添加NAD和谷氨酸作为共底物时，大肠杆菌的多粘菌素抗性突变体的提取物催化[α-P-32] UDP-葡萄糖醛酸的C-4”氧化和C-6”脱羧，然后转氨生成[α-P-32]UDP-L-Ara 4 N。此外，当包括N-10-甲酰四氢叶酸时，[α-（32）p]UDP-L-Ara 4 N被甲酰化。这些活动与pmrF操纵子的两种基因产物（PmrI和PmrH）的拟议功能一致。使用T7构建体过表达PmrI（重新命名为ArnA），并显示其本身催化UDP-葡萄糖醛酸的前所未有的氧化脱羧作用，形成尿苷5 '-（β-L-苏型-五吡喃糖基-4”-二磷酸酮糖）。将后者的6 mg样品纯化，并通过NMR研究验证其结构为4”酮的水合物。ArnA在氧化其底物的C-4”位置方面类似于UDP-半乳糖差向异构酶、dTDP-葡萄糖-4，6-脱氢酶和UDP-木糖合酶，但不同之处在于其释放NADH产物。
Addition of the 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiety to the phosphate groups of lipid A is implicated in bacterial resistance to polymyxin and cationic antimicrobial peptides of the innate immune system. The sequences of the products of the Salmonella typhimurium pmrE andpmrF loci, both of which are required for polymyxin resistance, recently led us to propose a pathway for L-Ara4N biosynthesis from UDP-glucuronic acid (Zhou, Z., Lin, S., Cotter, R. J., and Raetz, C. R. H. (1999) J. Biol. Chem. 274, 18503-18514). We now report that extracts of a polymyxin-resistant mutant of Escherichia coli catalyze the C-4" oxidation and C-6" decarboxylation of [alpha-P-32]UDP-glucuronic acid, followed by transamination to generate [alpha-P-32]UDP-L-Ara4N, when NAD and glutamate are added as co-substrates. In addition, the [alpha-(32)p]UDP-L-Ara4N is formylated when N-10-formyltetrahydrofolate is included. These activities are consistent with the proposed functions of two of the gene products (PmrI and PmrH) of the pmrF operon. PmrI (renamed ArnA) was overexpressed using a T7 construct, and shown by itself to catalyze the unprecedented oxidative decarboxylation of UDP-glucuronic acid to form uridine 5'-(beta-L-threo-pentapyranosyl-4"-ulose diphosphate). A 6-mg sample of the latter was purified, and its structure was validated by NMR studies as the hydrate of the 4" ketone. ArnA resembles UDP-galactose epimerase, dTDP-glucose-4,6-dehydratase, and UDP-xylose synthase in oxidizing the C-4" position of its substrate, but differs in that it releases the NADH product.