Department of Biochemistry, University of Wisconsin, Madison, Wisconsin, 53706.
Protein Sci. 2018 Mar;27(3):738-749. doi: 10.1002/pro.3368. Epub 2018 Feb 5.
Bacillus thuringiensis is a soil-dwelling Gram positive bacterium that has been utilized as a biopesticide for well over 60 years. It is known to contain flagella that are important for motility. One of the proteins found in flagella is flagellin, which is post-translationally modified by O-glycosylation with derivatives of pseudaminic acid. The biosynthetic pathway for the production of CMP-pseudaminic acid in B. thuringiensis, starting with UDP-N-acetyl-d-glucosamine (UDP-GlcNAc), requires seven enzymes. Here, we report the three-dimensional structures of Pen and Pal, which catalyze the first and second steps, respectively. Pen contains a tightly bound NADP(H) cofactor whereas Pal is isolated with bound NAD(H). For the X-ray analysis of Pen, the site-directed D128N/K129A mutant variant was prepared in order to trap its substrate, UDP-GlcNAc, into the active site. Pen adopts a hexameric quaternary structure with each subunit showing the bilobal architecture observed for members of the short-chain dehydrogenase/reductase superfamily. The hexameric quaternary structure is atypical for most members of the superfamily. The structure of Pal was determined in the presence of UDP. Pal adopts the more typical dimeric quaternary structure. Taken together, Pen and Pal catalyze the conversion of UDP-GlcNAc to UDP-4-keto-6-deoxy-l-N-acetylaltrosamine. Strikingly, in Gram negative bacteria such as Campylobacter jejuni and Helicobacter pylori, only a single enzyme (FlaA1) is required for the production of UDP-4-keto-6-deoxy-l-N-acetylaltrosamine. A comparison of Pen and Pal with FlaA1 reveals differences that may explain why FlaA1 is a bifunctional enzyme whereas Pen and Pal catalyze the individual steps leading to the formation of the UDP-sugar product. This investigation represents the first structural analysis of the enzymes in B. thuringiensis that are required for CMP-pseudaminic acid formation.
苏云金芽孢杆菌是一种革兰氏阳性土壤细菌，作为生物农药已经使用了超过 60 年。它被认为含有鞭毛，鞭毛对于运动性很重要。在鞭毛中发现的一种蛋白质是鞭毛蛋白，它通过 O-糖基化被假氨基糖酸的衍生物修饰。苏云金芽孢杆菌中 CMP-假氨基糖酸的生物合成途径，从 UDP-N-乙酰-d-葡萄糖胺（UDP-GlcNAc）开始，需要七种酶。在这里，我们报告了分别催化第一步和第二步的 Pen 和 Pal 的三维结构。Pen 含有紧密结合的 NADP（H）辅因子，而 Pal 则与结合的 NAD（H）一起分离。为了对 Pen 进行 X 射线分析，制备了定点突变的 D128N/K129A 变体，以便将其底物 UDP-GlcNAc 捕获到活性位点中。Pen 采用六聚体的四级结构，每个亚基呈现出短链脱氢酶/还原酶超家族成员观察到的双叶结构。这种六聚体的四级结构对于该超家族的大多数成员来说是非典型的。Pal 的结构在 UDP 的存在下被确定。Pal 采用更典型的二聚体四级结构。总的来说，Pen 和 Pal 催化 UDP-GlcNAc 向 UDP-4-酮-6-脱氧-l-N-乙酰-altrosamine 的转化。引人注目的是，在弯曲杆菌和幽门螺杆菌等革兰氏阴性细菌中，只需要一种酶（FlaA1）就可以产生 UDP-4-酮-6-脱氧-l-N-乙酰-altrosamine。Pen 和 Pal 与 FlaA1 的比较揭示了差异，这些差异可能解释了为什么 FlaA1 是一种多功能酶，而 Pen 和 Pal 催化形成 UDP-糖产物的各个步骤。这项研究代表了对苏云金芽孢杆菌中 CMP-假氨基糖酸形成所需的酶的首次结构分析。
