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Structural and Biochemical Investigation of PglF from Campylobacter jejuni Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily
Biochemistry ( IF 2.9 ) Pub Date : 2017-11-03 00:00:00 , DOI: 10.1021/acs.biochem.7b00910
Alexander S. Riegert 1 , James B. Thoden 1 , Ian C. Schoenhofen 2 , David C. Watson 2 , N. Martin Young 2 , Peter A. Tipton 3 , Hazel M. Holden 1
Affiliation  

Within recent years it has become apparent that protein glycosylation is not limited to eukaryotes. Indeed, in Campylobacter jejuni, a Gram-negative bacterium, more than 60 of its proteins are known to be glycosylated. One of the sugars found in such glycosylated proteins is 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, hereafter referred to as QuiNAc4NAc. The pathway for its biosynthesis, initiating with UDP-GlcNAc, requires three enzymes referred to as PglF, PglE, and PlgD. The focus of this investigation is on PglF, an NAD+-dependent sugar 4,6-dehydratase known to belong to the short chain dehydrogenase/reductase (SDR) superfamily. Specifically, PglF catalyzes the first step in the pathway, namely, the dehydration of UDP-GlcNAc to UDP-2-acetamido-2,6-dideoxy-α-d-xylo-hexos-4-ulose. Most members of the SDR superfamily contain a characteristic signature sequence of YXXXK where the conserved tyrosine functions as a catalytic acid or a base. Strikingly, in PglF, this residue is a methionine. Here we describe a detailed structural and functional investigation of PglF from C. jejuni. For this investigation five X-ray structures were determined to resolutions of 2.0 Å or better. In addition, kinetic analyses of the wild-type and site-directed variants were performed. On the basis of the data reported herein, a new catalytic mechanism for a SDR superfamily member is proposed that does not require the typically conserved tyrosine residue.

中文翻译:

空肠弯曲杆菌PglF的结构和生化研究揭示了短链脱氢酶/还原酶超家族成员的新机制

近年来,蛋白质糖基化不限于真核生物已变得显而易见。确实,在空肠弯曲菌中,革兰氏阴性细菌的蛋白质中有60多种被糖基化。在这种糖基化的蛋白质中发现的糖之一是2,4-二乙酰氨基-2,4,6-三苯氧基-α - d-吡喃葡萄糖,以下称为Quinacc4NAc。从UDP-GlcNAc开始的生物合成途径需要三种酶,分别称为PglF,PglE和PlgD。这项调查的重点是PglF,NAD +依赖性糖4,6-脱水酶属于短链脱氢酶/还原酶(SDR)超家族。具体而言,PglF催化了该途径的第一步,即UDP-GlcNAc脱水为UDP-2-乙酰氨基-2,6-二脱氧-α- d-木糖-己糖-4-ulose。SDR超家族的大多数成员都包含YXXXK的特征性签名序列,其中保守的酪氨酸起着催化酸或碱的作用。引人注目的是,在PglF中,该残基是蛋氨酸。在这里,我们描述了来自空肠弯曲菌的PglF的详细结构和功能研究。为了进行这项研究,确定了五个X射线结构,其分辨率为2.0Å或更高。此外,还对野生型和定点变异进行了动力学分析。基于本文报道的数据,提出了不需要特别通常保守的酪氨酸残基的SDR超家族成员的新催化机制。
更新日期:2017-11-03
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