Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan; Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan.
J Struct Biol. 2011 Sep;175(3):300-10. doi: 10.1016/j.jsb.2011.04.010. Epub 2011 Apr 23.
Cationic modification of lipid A with 4-amino-4-deoxy-L-arabinopyranose (L-Ara4N) allows the pathogen Klebsiella pneumoniae to resist the antibiotic polymyxin and other cationic antimicrobial peptides. UDP-glucose dehydrogenase (Ugd) catalyzes the NAD⁺-dependent twofold oxidation of UDP-glucose (UPG) to produce UDP-glucuronic acid (UGA), a requisite precursor in the biosynthesis of L-Ara4N and bacterial exopolysaccharides. Here we report five crystal structures of K. pneumoniae Ugd (KpUgd) in its apo form, in complex with UPG, UPG/NADH, two UGA molecules, and finally with a C-terminal His₆-tag. The UGA-complex structure differs from the others by a 14° rotation of the N-terminal domain toward the C-terminal domain, and represents a closed enzyme conformation. It also reveals that the second UGA molecule binds to a pre-existing positively charged surface patch away from the active site. The enzyme is thus inactivated by moving the catalytically important residues C253, K256 and D257 from their original positions. Kinetic data also suggest that KpUgd has multiple binding sites for UPG, and that UGA is a competitive inhibitor. The conformational changes triggered by UGA binding to the allosteric site can be exploited in designing potent inhibitors.
脂多糖的阳离子修饰与 4-氨基-4-去氧-L-阿拉伯吡喃糖（L-Ara4N）结合，使病原体肺炎克雷伯氏菌能够抵抗抗生素多粘菌素和其他阳离子抗菌肽。UDP-葡萄糖脱氢酶（Ugd）催化 NAD⁺依赖性二倍体氧化 UDP-葡萄糖（UPG）产生 UDP-葡萄糖醛酸（UGA），这是 L-Ara4N 和细菌胞外多糖生物合成的必需前体。在这里，我们报告了肺炎克雷伯氏菌 Ugd（KpUgd）在apo 形式、与 UPG、UPG/NADH、两个 UGA 分子以及最后与 C 末端 His₆ 标签的复合物的五个晶体结构。UGA 复合物结构与其他结构的区别在于 N 末端结构域相对于 C 末端结构域旋转了 14°，代表了一种封闭的酶构象。它还揭示了第二个 UGA 分子结合到远离活性部位的预先存在的正电荷表面斑块上。因此，通过将催化重要残基 C253、K256 和 D257 从其原始位置移动，酶失活。动力学数据还表明，KpUgd 对 UPG 具有多个结合位点，而 UGA 是竞争性抑制剂。UGA 结合到别构位点引发的构象变化可用于设计有效的抑制剂。
