Galactarate dehydratase (GarD) is the first enzyme in the galactarate/glucarate pathway and catalyzes the dehydration of galactarate to 3-keto-5-dehydroxygalactarate. This protein is known to increase colonization fitness of intestinal pathogens in antibiotic-treated mice and to promote bacterial survival during stress. The galactarate/glucarate pathway is widespread in bacteria, but not in humans, and thus could be a target to develop new inhibitors for use in combination therapy to combat antibiotic resistance. The structure of almost all the enzymes of the galactarate/glucarate pathway were solved previously, except for GarD, for which only the structure of the N-terminal domain was determined previously. Herein, we report the first crystal structure of full-length GarD solved using a seleno-methoionine derivative revealing a new protein fold. The protein consists of three domains, each presenting a novel twist as compared to their distant homologs. GarD in the crystal structure forms dimers and each monomer consists of three domains. The N-terminal domain is comprised of a β-clip fold, connected to the second domain by a long unstructured linker. The second domain serves as a dimerization interface between two monomers. The C-terminal domain forms an unusual variant of a Rossmann fold with a crossover and is built around a seven-stranded parallel β-sheet supported by nine α-helices. A metal binding site in the C-terminal domain is occupied by Ca2+ . The activity of GarD was corroborated by the production of 5-keto-4-deoxy-D-glucarate under reducing conditions and in the presence of iron. Thus, GarD is an unusual enolase with a novel protein fold never previously seen in this class of enzymes.

中文翻译：

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半乳糖二酸脱水酶的结构，烯醇酶的一种新折叠，参与抗生素治疗后细菌的适应性。


半乳糖二酸脱水酶 (GarD) 是半乳糖二酸/葡萄糖二酸途径中的第一个酶，催化半乳糖二酸脱水为 3-酮-5-脱羟基半乳糖二酸。众所周知，这种蛋白质可以增加抗生素治疗小鼠肠道病原体的定植适应性，并促进压力期间细菌的存活。半乳糖二酸/葡萄糖二酸途径在细菌中广泛存在，但在人类中并不普遍，因此可能成为开发新抑制剂用于联合治疗以对抗抗生素耐药性的目标。半乳糖二酸/葡萄糖二酸途径的几乎所有酶的结构均已在之前得到解决，但GarD除外，之前仅确定了GarD的N端结构域的结构。在此，我们报告了使用硒代甲硫氨酸衍生物解析的全长 GarD 的第一个晶体结构，揭示了新的蛋白质折叠。该蛋白质由三个结构域组成，每个结构域与其遥远的同源物相比都呈现出新颖的变化。 GarD在晶体结构中形成二聚体，每个单体由三个结构域组成。 N 端结构域由 β 夹折叠组成，通过长的非结构化接头连接到第二个结构域。第二个结构域充当两个单体之间的二聚化界面。 C 末端结构域形成罗斯曼折叠的一种不寻常的变体，具有交叉，并围绕由九个 α 螺旋支撑的七链平行 β 折叠构建。 C 端结构域中的金属结合位点被 Ca2+ 占据。 GarD 的活性通过在还原条件下和铁存在下产生 5-酮-4-脱氧-D-葡萄糖二酸得到证实。因此，GarD 是一种不寻常的烯醇化酶，具有此类酶中从未见过的新颖蛋白质折叠。