Alphey Magnus S, Pirrie Lisa, Torrie Leah S, Boulkeroua Wassila Abdelli, Gardiner Mary, Sarkar Aurijit, Maringer Marko, Oehlmann Wulf, Brenk Ruth, Scherman Michael S, McNeil Michael, Rejzek Martin, Field Robert A, Singh Mahavir, Gray David, Westwood Nicholas J, Naismith James H
Biomedical Sciences Research Complex, University of St. Andrews , St. Andrews KY16 9ST, U.K.
ACS Chem Biol. 2013 Feb 15;8(2):387-96. doi: 10.1021/cb300426u. Epub 2012 Nov 28.
Glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the condensation of glucose-1-phosphate (G1P) with deoxy-thymidine triphosphate (dTTP) to yield dTDP-d-glucose and pyrophosphate. This is the first step in the l-rhamnose biosynthetic pathway. l-Rhamnose is an important component of the cell wall of many microorganisms, including Mycobacterium tuberculosis and Pseudomonas aeruginosa. Here we describe the first nanomolar inhibitors of P. aeruginosa RmlA. These thymine analogues were identified by high-throughput screening and subsequently optimized by a combination of protein crystallography, in silico screening, and synthetic chemistry. Some of the inhibitors show inhibitory activity against M. tuberculosis. The inhibitors do not bind at the active site of RmlA but bind at a second site remote from the active site. Despite this, the compounds act as competitive inhibitors of G1P but with high cooperativity. This novel behavior was probed by structural analysis, which suggests that the inhibitors work by preventing RmlA from undergoing the conformational change key to its ordered bi-bi mechanism.
葡萄糖-1-磷酸胸苷酰转移酶（RmlA）催化葡萄糖-1-磷酸（G1P）与脱氧胸苷三磷酸（dTTP）缩合，生成dTDP-d-葡萄糖和焦磷酸。这是L-鼠李糖生物合成途径的第一步。L-鼠李糖是许多微生物细胞壁的重要组成部分，包括结核分枝杆菌和铜绿假单胞菌。在此，我们描述了铜绿假单胞菌RmlA的首个纳摩尔级抑制剂。这些胸腺嘧啶类似物通过高通量筛选鉴定，随后通过蛋白质晶体学、计算机模拟筛选和合成化学相结合的方法进行优化。一些抑制剂对结核分枝杆菌具有抑制活性。这些抑制剂并非结合在RmlA的活性位点，而是结合在远离活性位点的第二个位点。尽管如此，这些化合物作为G1P的竞争性抑制剂，但具有高协同性。通过结构分析对这种新行为进行了探究，结果表明抑制剂的作用方式是阻止RmlA发生对其有序双底物双产物机制至关重要的构象变化。
