Department of Chemistry , Texas A&M University , College Station , Texas 77843 , United States.
Department of Biochemistry , University of Wisconsin-Madison , Madison , Wisconsin 53706 , United States.
Biochemistry. 2019 Aug 6;58(31):3340-3353. doi: 10.1021/acs.biochem.9b00326. Epub 2019 Jul 26.
The gene cluster is found in 80% of sequenced genomes and other closely related species in the human microbiome. On the basis of the annotations of the enzymes located in this cluster, it is expected that together they catalyze the catabolism of an unknown carbohydrate. The focus of this investigation is on YdjI, which is in the gene cluster of K-12. It is predicted to be a class II aldolase of unknown function. Here we describe a structural and functional characterization of this enzyme. YdjI catalyzes the hydrogen/deuterium exchange of the pro- hydrogen at C3 of dihydroxyacetone phosphate (DHAP). In the presence of DHAP, YdjI catalyzes an aldol condensation with a variety of aldo sugars. YdjI shows a strong preference for higher-order (seven-, eight-, and nine-carbon) monosaccharides with specific hydroxyl stereochemistries and a negatively charged terminus (carboxylate or phosphate). The best substrate is l-arabinuronic acid with an apparent of 3.0 s. The product, l--l--octuluronate-1-phosphate, has a / value of 2.1 × 10 M s in the retro-aldol reaction with YdjI. This is the first recorded synthesis of l--l--octuluronate-1-phosphate and six similar carbohydrates. The crystal structure of YdjI, determined to a nominal resolution of 1.75 Å (Protein Data Bank entry 6OFU ), reveals unusual positions for two arginine residues located near the active site. Computational docking was utilized to distinguish preferable binding orientations for l--l--octuluronate-1-phosphate. These results indicate a possible alternative binding orientation for l--l--octuluronate-1-phosphate compared to that observed in other class II aldolases, which utilize shorter carbohydrate molecules.
该基因簇存在于 80%已测序的基因组和人类微生物组中其他密切相关的物种中。根据位于该簇中的酶的注释，可以预期它们共同催化未知碳水化合物的分解代谢。本研究的重点是 K-12 中的基因簇中的 YdjI。它预计是一种未知功能的 II 类醛缩酶。在这里，我们描述了该酶的结构和功能特征。YdjI 催化二羟丙酮磷酸（DHAP）的 C3 上的质子/氘交换。在 DHAP 的存在下，YdjI 催化各种醛糖的醛缩合。YdjI 对具有特定羟基立体化学和带负电荷末端（羧基或磷酸基）的较高阶（七、八和九碳）单糖表现出强烈的偏好。最佳底物是 l-阿拉伯糖酸，表观值为 3.0 s。产物 l--l--辛糖-1-磷酸与 YdjI 的 retro-aldol 反应的 值为 2.1×10 M s。这是首次记录的 l--l--辛糖-1-磷酸和六种类似碳水化合物的合成。YdjI 的晶体结构，确定的名义分辨率为 1.75 Å（蛋白质数据库条目 6OFU），揭示了靠近活性位点的两个精氨酸残基的异常位置。计算对接用于区分 l--l--辛糖-1-磷酸的优选结合取向。这些结果表明与其他 II 类醛缩酶相比，l--l--辛糖-1-磷酸可能具有替代的结合取向，后者利用较短的碳水化合物分子。
