Structure and function of both domains of ArnA, a dual function decarboxylase and a formyltransferase, involved in 4-amino-4-deoxy-L-arabinose biosynthesis
Structure and function of both domains of ArnA, a dual function decarboxylase and a formyltransferase, involved in 4-amino-4-deoxy-L-arabinose biosynthesis
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DOI：
10.1074/jbc.m501534200
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发表时间：
2005-06-17
影响因子：
4.8
通讯作者：
Naismith, JH
中科院分区：
文献类型：
--
作者：
Williams, GJ;Breazeale, SD;Naismith, JH
通过添加糖4-氨基-4-脱氧-L-阿拉伯糖（L-Ara 4 N）来修饰脂多糖的脂质A部分是致病性革兰氏阴性菌逃避先天免疫系统产生的阳离子抗微生物肽的策略。因此，L-Ara 4 N生物合成是一个潜在的抗感染靶点，因为抑制其合成会使某些病原体对免疫系统更敏感。L-Ara 4 N生物合成所需的双功能酶ArnA催化UDP-葡糖醛酸的NAD（+）依赖性氧化脱羧以产生UDP-4 '-酮戊糖，并且还催化甲酰基从N-10-甲酰基四氢叶酸转移到UDP-L-Ara 4 N的4'-胺。我们现在报告的晶体结构的N-末端甲酰转移酶结构域与尿苷一磷酸和N-5-甲酰四氢叶酸的复合物。利用这种结构，我们确定了ArnA中甲酰转移的活性位点，包括关键的催化残基Asn（102），His（104）和Asp（140）。此外，我们已经证明脱羧酶结构域的残基Ser（433）和Glu（434）是UDP-GlcUA氧化脱羧所必需的.一个E434 Q突变体是无活性的，这表明该残基的化学性质而不是空间性质在脱羧反应中至关重要。我们的数据表明，脱羧酶结构域催化氢化物提取（氧化）从C-4'位置和随后的脱羧。
Modification of the lipid A moiety of lipopolysaccharide by the addition of the sugar 4-amino-4-deoxy-L-arabinose (L-Ara4N) is a strategy adopted by pathogenic Gram-negative bacteria to evade cationic antimicrobial peptides produced by the innate immune system. L-Ara4N biosynthesis is therefore a potential anti-infective target, because inhibiting its synthesis would render certain pathogens more sensitive to the immune system. The bifunctional enzyme ArnA, which is required for L-Ara4N biosynthesis, catalyzes the NAD(+)-dependent oxidative decarboxylation of UDP-glucuronic acid to generate a UDP-4'-keto-pentose sugar and also catalyzes transfer of a formyl group from N-10-formyltetrahydrofolate to the 4'-amine of UDP-L-Ara4N. We now report the crystal structure of the N-terminal formyltransferase domain in a complex with uridine monophosphate and N-5-formyltetrahydrofolate. Using this structure, we identify the active site of formyltransfer in ArnA, including the key catalytic residues Asn(102), His(104), and Asp(140). Additionally, we have shown that residues Ser(433) and Glu(434) of the decarboxylase domain are required for the oxidative decarboxylation of UDP- GlcUA. An E434Q mutant is inactive, suggesting that chemical rather than steric properties of this residue are crucial in the decarboxylation reaction. Our data suggest that the decarboxylase domain catalyzes both hydride abstraction (oxidation) from the C-4' position and the subsequent decarboxylation.