Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan.
Structural Biology Research Center, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Japan.
FEBS J. 2018 Apr;285(8):1540-1555. doi: 10.1111/febs.14429. Epub 2018 Mar 24.
Enzymes belonging to the aspartase/fumarase superfamily catalyze elimination of various functional groups from succinate derivatives and play an important role in primary metabolism and aromatic compound degradation. Recently, an aspartase/fumarase superfamily enzyme, CreD, was discovered in cremeomycin biosynthesis. This enzyme catalyzes the elimination of nitrous acid from nitrosuccinate synthesized from aspartate by CreE, a flavin-dependent monooxygenase. Nitrous acid generated by this pathway is an important precursor of the diazo group of cremeomycin. CreD is the first aspartase/fumarase superfamily enzyme that was reported to catalyze the elimination of nitrous acid, and therefore we aimed to analyze its reaction mechanism. The crystal structure of CreD was determined by the molecular replacement native-single anomalous diffraction method at 2.18 Å resolution. Subsequently, the CreD-fumarate complex structure was determined at 2.30 Å resolution by the soaking method. Similar to other aspartase/fumarase superfamily enzymes, the crystal structure of CreD was composed of three domains and formed a tetramer. Two molecules of fumarate were observed in one subunit of the CreD-fumarate complex. One of them was located in the active site pocket formed by three different subunits. Intriguingly, no histidine residue, which usually functions as a catalytic acid in aspartase/fumarase superfamily enzymes, was found around the fumarate molecule in the active site. Based on the mutational analysis, we propose a catalytic mechanism of CreD, in which Arg325 acts as a catalytic acid.
The crystal structures of CreD and the CreD-fumarate complex were deposited to PDB under the accession numbers 5XNY and 5XNZ, respectively.
Nitrosuccinate lyase CreD, EC4.3.
属于天冬氨酸酶/富马酸酶超家族的酶催化各种功能基团从琥珀酸衍生物中消除，在初级代谢和芳香族化合物降解中发挥重要作用。最近，在cremeomycin 生物合成中发现了一种天冬氨酸酶/富马酸酶超家族酶 CreD。该酶催化由 CreE（一种黄素依赖性单加氧酶）合成的天冬氨酸衍生的亚硝基琥珀酸盐中亚硝酸的消除。该途径产生的亚硝酸是 cremeomycin 的重氮基团的重要前体。CreD 是第一个报道催化亚硝酸消除的天冬氨酸酶/富马酸酶超家族酶，因此我们旨在分析其反应机制。通过分子置换原生单异常衍射方法，以 2.18Å 的分辨率确定 CreD 的晶体结构。随后，通过浸泡法以 2.30Å 的分辨率确定 CreD-富马酸盐复合物的结构。与其他天冬氨酸酶/富马酸酶超家族酶一样，CreD 的晶体结构由三个结构域组成，并形成四聚体。在 CreD-富马酸盐复合物的一个亚基中观察到两个富马酸盐分子。其中一个位于由三个不同亚基形成的活性位点口袋中。有趣的是，在活性位点中没有发现组氨酸残基，组氨酸残基通常在天冬氨酸酶/富马酸酶超家族酶中作为催化酸发挥作用。基于突变分析，我们提出了 CreD 的催化机制，其中 Arg325 作为催化酸。
CreD 和 CreD-富马酸盐复合物的晶体结构分别以 5XNY 和 5XNZ 的登录号提交到 PDB。
亚硝基琥珀酸盐裂解酶 CreD，EC4.3。
