Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Biochemistry. 2010 Aug 24;49(33):7227-37. doi: 10.1021/bi100805b.
In recent years, the opportunistic pathogen Pseudomonas aeruginosa has emerged as a major source of hospital-acquired infections. Effective treatment has proven increasingly difficult due to the spread of multidrug resistant strains and thus requires a deeper understanding of the biochemical mechanisms of pathogenicity. The central carbohydrate of the P. aeruginosa PAO1 (O5) B-band O-antigen, ManNAc(3NAc)A, has been shown to be critical for virulence and is produced in a stepwise manner by five enzymes in the Wbp pathway (WbpA, WbpB, WbpE, WbpD, and WbpI). Herein, we present the crystal structure of the aminotransferase WbpE from P. aeruginosa PAO1 in complex with the cofactor pyridoxal 5'-phosphate (PLP) and product UDP-GlcNAc(3NH(2))A as the external aldimine at 1.9 A resolution. We also report the structures of WbpE in complex with PMP alone as well as the PLP internal aldimine and show that the dimeric structure of WbpE observed in the crystal structure is confirmed by analytical ultracentrifugation. Analysis of these structures reveals that the active site of the enzyme is composed of residues from both subunits. In particular, we show that a key residue (Arg229), which has previously been implicated in direct interactions with the alpha-carboxylate moiety of alpha-ketoglutarate, is also uniquely positioned to bestow specificity for the 6''-carboxyl group of GlcNAc(3NH(2))A through a salt bridge. This finding is intriguing because while an analogous basic residue is present in WbpE homologues that do not process 6''-carboxyl-modified saccharides, recent structural studies reveal that this side chain is retracted to accommodate a neutral C6'' atom. This work represents the first structural analysis of a nucleotide sugar aminotransferase with a bound product modified at the C2'', C3'', and C6'' positions and provides insight into a novel target for treatment of P. aeruginosa infection.
近年来，机会性病原体铜绿假单胞菌已成为医院获得性感染的主要来源。由于多药耐药株的传播，有效的治疗变得越来越困难，因此需要更深入地了解致病性的生化机制。铜绿假单胞菌 PAO1（O5）B 带 O-抗原的核心碳水化合物 ManNAc(3NAc)A 已被证明对毒力至关重要，并且通过 Wbp 途径中的五种酶（WbpA、WbpB、WbpE、WbpD 和 WbpI）以逐步方式产生。在此，我们呈现了铜绿假单胞菌 PAO1 中的氨基转移酶 WbpE 与辅因子吡哆醛 5'-磷酸（PLP）和产物 UDP-GlcNAc(3NH(2))A 作为外部醛亚胺的复合物的晶体结构，分辨率为 1.9A。我们还报告了 WbpE 与 PMP 单独复合物以及 PLP 内部醛亚胺的结构，并表明在晶体结构中观察到的 WbpE 二聚体结构通过分析超速离心得到证实。这些结构的分析表明，该酶的活性位点由两个亚基的残基组成。特别是，我们表明，以前被认为与α-酮戊二酸的α-羧基部分直接相互作用的关键残基（Arg229）也通过盐桥独特地定位于赋予 GlcNAc(3NH(2))A 的 6''-羧基特异性。这一发现很有趣，因为虽然在不处理 6''-羧基修饰糖的 WbpE 同源物中存在类似的碱性残基，但最近的结构研究表明，该侧链回缩以适应中性 C6''原子。这项工作代表了第一个与结合产物在 C2''、C3''和 C6''位置修饰的核苷酸糖氨基转移酶的结构分析，并为治疗铜绿假单胞菌感染的新靶标提供了深入了解。
