Department of Biochemistry, University of Colorado Boulder, Boulder, Colorado 80309, United States.
Department of Chemistry, University of Colorado Boulder, Boulder, Colorado 80309, United States.
Biochemistry. 2023 Jul 18;62(14):2216-2227. doi: 10.1021/acs.biochem.3c00227. Epub 2023 Jul 6.
Polymyxins are important last resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. However, pathogens have acquired resistance to polymyxins through a pathway that modifies lipid A with 4-amino-4-deoxy-l-arabinose (Ara4N). Inhibition of this pathway is, therefore, a desirable strategy to combat polymyxin resistance. The first pathway-specific reaction is an NAD-dependent oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcA) catalyzed by the dehydrogenase domain of ArnA (ArnA_DH). We present the crystal structure of serovar typhimurium ArnA in complex with UDP-GlcA showing that binding of the sugar nucleotide is sufficient to trigger a conformational change conserved in bacterial ArnA_DHs but absent in its human homologs, as confirmed by structure and sequence analysis. Ligand binding assays show that the conformational change is essential for NAD binding and catalysis. Enzyme activity and binding assays show that (i) UDP-GlcA analogs lacking the 6' carboxylic acid bind the enzyme but fail to trigger the conformational change, resulting in poor inhibition, and (ii) the uridine monophosphate moiety of the substrate provides most of the ligand binding energy. Mutation of asparagine 492 to alanine (N492A) disrupts the ability of ArnA_DH to undergo the conformational change while retaining substrate binding, suggesting that N492 is involved in sensing the 6' carboxylate in the substrate. These results identify the UDP-GlcA-induced conformational change in ArnA_DH as an essential mechanistic step in bacterial enzymes, providing a platform for selective inhibition.
多黏菌素是治疗多重耐药革兰氏阴性病原体感染的重要最后手段抗生素。然而，病原体通过修饰脂质 A 上的 4-氨基-4-脱氧-l-阿拉伯糖（Ara4N）获得了对多黏菌素的耐药性。因此，抑制这种途径是对抗多黏菌素耐药性的一种理想策略。该途径的第一个特异性反应是由 ArnA 的脱氢酶结构域（ArnA_DH）催化的 NAD 依赖性 UDP-葡萄糖醛酸（UDP-GlcA）氧化脱羧反应。我们展示了 serovar typhimurium ArnA 与 UDP-GlcA 复合物的晶体结构，表明糖核苷酸的结合足以触发细菌 ArnA_DHs 中保守但在其人类同源物中不存在的构象变化，这一点通过结构和序列分析得到了证实。配体结合实验表明，构象变化对于 NAD 结合和催化至关重要。酶活性和结合实验表明，（i）缺乏 6'羧酸的 UDP-GlcA 类似物结合酶但不能触发构象变化，导致抑制作用不佳，以及（ii）底物的尿嘧啶单磷酸部分提供了大部分配体结合能。将天冬酰胺 492 突变为丙氨酸（N492A）破坏了 ArnA_DH 发生构象变化的能力，同时保留了底物结合，这表明 N492 参与了对底物中 6'羧酸盐的感应。这些结果确定了 ArnA_DH 中 UDP-GlcA 诱导的构象变化是细菌酶中的一个必要的机制步骤，为选择性抑制提供了一个平台。
