Lee Myeongseon, Sousa Marcelo C
Department of Chemistry and Biochemistry, University of Colorado at Boulder , Boulder, Colorado 80309-0596, United States.
Biochemistry. 2014 Feb 4;53(4):796-805. doi: 10.1021/bi4015677. Epub 2014 Jan 24.
Cationic Antimicrobial Peptides (CAMPs) represent a first line of defense against bacterial colonization. When fighting Gram-negative bacteria, CAMPs initially interact electrostatically with the negatively charged phosphate groups in lipid A and are thought to kill bacteria by disrupting their membrane integrity. However, many human pathogens, including Salmonella and Pseudomonas , have evolved lipid A modification mechanisms that result in resistance to CAMPs and related antibiotics such as Colistin. The addition of 4-amino-4-deoxy-l-Arabinose (Ara4N) to a phosphate group in lipid A is one such modification, frequently found in Pseudomonas isolated from cystic fibrosis patients. The pathway for biosynthesis of Ara4N-lipid A requires conversion of UDP-Glucuronic acid into UDP-Ara4N and subsequent transfer of the amino-sugar to lipid A. ArnB is a pyridoxal-phosphate (PLP) dependent transaminase that catalyzes a crucial step in the pathway: synthesis of UDP-Ara4N from UDP-4-keto-pentose. Here we present the 2.3 Å resolution crystal structure of an active site mutant of ArnB (K188A) in complex with the reaction intermediate aldimine formed by UDP-Ara4N and PLP. The sugar-nucleotide binding site is in a cleft between the subunits of the ArnB dimer with the uracil buried at the interface and the UDP ribose and phosphate groups exposed to the solvent. The Ara4N moiety is found in the (4)C1 conformation and its positioning, stabilized by interactions with both the protein and cofactor, is compatible with catalysis. The structure suggests strategies for the development of specific inhibitors that may prove useful in the treatment of resistant bacteria such as Pseudomonas found in cystic fibrosis patients.
阳离子抗菌肽（CAMPs）是抵御细菌定植的第一道防线。在对抗革兰氏阴性菌时，CAMPs最初通过静电作用与脂多糖A中带负电荷的磷酸基团相互作用，并被认为通过破坏细菌的膜完整性来杀死细菌。然而，许多人类病原体，包括沙门氏菌和铜绿假单胞菌，已经进化出脂多糖A修饰机制，从而对CAMPs和相关抗生素如多粘菌素产生抗性。在脂多糖A的磷酸基团上添加4-氨基-4-脱氧-L-阿拉伯糖（Ara4N）就是这样一种修饰，在从囊性纤维化患者中分离出的铜绿假单胞菌中经常发现。Ara4N-脂多糖A的生物合成途径需要将UDP-葡萄糖醛酸转化为UDP-Ara4N，随后将氨基糖转移到脂多糖A上。ArnB是一种依赖磷酸吡哆醛（PLP）的转氨酶，它催化该途径中的一个关键步骤：从UDP-4-酮戊糖合成UDP-Ara4N。在这里，我们展示了ArnB活性位点突变体（K188A）与由UDP-Ara4N和PLP形成的反应中间体醛亚胺复合物的2.3 Å分辨率晶体结构。糖核苷酸结合位点位于ArnB二聚体亚基之间的裂隙中，尿嘧啶埋在界面处，UDP核糖和磷酸基团暴露于溶剂中。Ara4N部分呈（4）C1构象，其定位通过与蛋白质和辅因子的相互作用得以稳定，这与催化作用相兼容。该结构为开发特异性抑制剂提供了策略，这些抑制剂可能对治疗囊性纤维化患者中发现的耐药细菌如铜绿假单胞菌有用。
