Analysis of the arabinose-5-phosphate isomerase of Bacteroides fragilis provides insight into regulation of single-domain arabinose phosphate isomerases.
Analysis of the arabinose-5-phosphate isomerase of Bacteroides fragilis provides insight into regulation of single-domain arabinose phosphate isomerases.
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对脆弱拟杆菌的阿拉伯糖 5-磷酸异构酶的分析提供了对单域阿拉伯糖磷酸异构酶调节的深入了解。
DOI：
10.1128/jb.01735-14
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发表时间：
2014
影响因子：
3.2
通讯作者：
Woodard,RonaldW
中科院分区：
文献类型：
--
作者：
Cech,David;Wang,PanFen;Holler,TodP;Woodard,RonaldW
阿拉伯糖-5-磷酸异构酶(APIs)催化D-核酮糖-5-磷酸和D-阿拉伯糖-5-磷酸的相互转化，是革兰氏阴性菌合成3-脱氧-D-甘露-辛酸(KDO)的第一步，3-脱氧-D-甘露-辛酸(KDO)是革兰氏阴性菌脂多糖的重要组成部分。经典的原料药，如大肠杆菌KdsD，含有一个糖异构酶结构域和一个串联的胱硫醚β-合成酶结构域。尽管付出了大量的努力，但人们对这些API中的结构-函数关系知之甚少。我们最近报道了一种只含有糖异构酶结构域的API。这种来自E.ColiCFT073的蛋白质C3406没有已知的生理功能。在这项研究中，我们研究了厌氧革兰氏阴性杆菌脆弱类杆菌的一个假定的单结构域API。这个推测的API(UniProt ID Q5LIW1)是脆弱芽孢杆菌基因组编码的唯一与任何已知API有显著同源性的蛋白质，表明它负责脆弱芽孢杆菌内毒素的生物合成。我们通过制备重组Q5LIW1蛋白(这里指的是UniProt ID Q5LIW1)，体外鉴定其API活性，并证明编码Q5LIW1的基因(GenBank ID YP_209877.1)能够补充API缺陷的大肠杆菌菌株，从而验证了这一假设。我们证明了Q5LIW1被Kdo生物合成途径的最终产物胞苷5‘-单磷-3-脱氧-d-甘露-2-辛酸抑制，Ki值为1.91μM。这些结果支持了Q5LIW1是支持脆弱芽孢杆菌内毒素生物合成的API，并受到CMPKdo的反馈调控。大肠杆菌KdsD的糖异构酶结构域缺乏两个半胱硫氨酸β-合成酶结构域，显示了API活性，并对其进行了进一步鉴定。这些结果表明，Q5LIW1可能是研究API结构-功能关系的合适系统。
Arabinose-5-phosphate isomerases (APIs) catalyze the interconversion ofd-ribulose-5-phosphate andd-arabinose-5-phosphate, the first step in the biosynthesis of 3-deoxy-d-manno-octulosonic acid (Kdo), an essential component of the lipopolysaccharide in Gram-negative bacteria. Classical APIs, such as Escherichia coli KdsD, contain a sugar isomerase domain and a tandem cystathionine beta-synthase domain. Despite substantial effort, little is known about structure-function relationships in these APIs. We recently reported an API containing only a sugar isomerase domain. This protein, c3406 from E. coli CFT073, has no known physiological function. In this study, we investigated a putative single-domain API from the anaerobic Gram-negative bacterium Bacteroides fragilis. This putative API (UniProt ID Q5LIW1) is the only protein encoded by the B. fragilis genome with significant identity to any known API, suggesting that it is responsible for lipopolysaccharide biosynthesis in B. fragilis. We tested this hypothesis by preparing recombinant Q5LIW1 protein (here referred to by the UniProt ID Q5LIW1), characterizing its API activityin vitro, and demonstrating that the gene encoding Q5LIW1 (GenBank ID YP_209877.1) was able to complement an API-deficient E. coli strain. We demonstrated that Q5LIW1 is inhibited by cytidine 5′-monophospho-3-deoxy-d-manno-2-octulosonic acid, the final product of the Kdo biosynthesis pathway, with aKiof 1.91 μM. These results support the assertion that Q5LIW1 is the API that supports lipopolysaccharide biosynthesis in B. fragilis and is subject to feedback regulation by CMP-Kdo. The sugar isomerase domain of E. coli KdsD, lacking the two cystathionine beta-synthase domains, demonstrated API activity and was further characterized. These results suggest that Q5LIW1 may be a suitable system to study API structure-function relationships.