Shah Bhumika S, Ashwood Heather E, Harrop Stephen J, Farrugia Daniel N, Paulsen Ian T, Mabbutt Bridget C
Department of Molecular Sciences, Macquarie University, Sydney, Australia.
School of Physics, The University of New South Wales, Sydney, Australia.
PLoS One. 2018 Jan 19;13(1):e0191610. doi: 10.1371/journal.pone.0191610. eCollection 2018.
With new strains of Acinetobacter baumannii undergoing genomic analysis, it has been possible to define regions of genomic plasticity (RGPs), encoding specific adaptive elements. For a selected RGP from a community-derived isolate of A. baumannii, we outline sequences compatible with biosynthetic machinery of surface polysaccharides, specifically enzymes utilized in the dehydration and conversion of UDP-N-acetyl-D-glucosamine (UDP-D-GlcNAc). We have determined the crystal structure of one of these, the epimerase Ab-WbjB. This dehydratase belongs to the 'extended' short-chain dehydrogenase/reductase (SDR) family, related in fold to previously characterised enzymes CapE and FlaA1. Our 2.65Å resolution structure of Ab-WbjB shows a hexamer, organised into a trimer of chain pairs, with coenzyme NADP+ occupying each chain. Specific active-site interactions between each coenzyme and a lysine quaternary group of a neighbouring chain interconnect adjacent dimers, so stabilising the hexameric form. We show UDP-GlcNAc to be a specific substrate for Ab-WbjB, with binding evident by ITC (Ka = 0.23 μmol-1). The sequence of Ab-WbjB shows variation from the consensus active-site motifs of many SDR enzymes, demonstrating a likely catalytic role for a specific threonine sidechain (as an alternative to tyrosine) in the canonical active site chemistry of these epimerases.
随着对鲍曼不动杆菌新菌株进行基因组分析，现已能够确定基因组可塑性区域（RGP），这些区域编码特定的适应性元件。对于从社区来源的鲍曼不动杆菌分离株中选择的一个RGP，我们概述了与表面多糖生物合成机制相容的序列，特别是参与UDP-N-乙酰-D-葡萄糖胺（UDP-D-GlcNAc）脱水和转化的酶。我们已经确定了其中一种酶——表异构酶Ab-WbjB的晶体结构。这种脱水酶属于“扩展”短链脱氢酶/还原酶（SDR）家族，其折叠结构与先前表征的CapE和FlaA1酶相关。我们解析得到的Ab-WbjB晶体结构分辨率为2.65Å，显示其为六聚体，由链对三聚体组成，每个链上都有辅酶NADP+。每个辅酶与相邻链的赖氨酸四级基团之间的特定活性位点相互作用将相邻二聚体相互连接，从而稳定六聚体形式。我们发现UDP-GlcNAc是Ab-WbjB的特异性底物，ITC实验表明其具有明显的结合作用（Ka = 0.23 μmol-1）。Ab-WbjB的序列与许多SDR酶的共有活性位点基序存在差异，这表明在这些表异构酶的典型活性位点化学中，特定的苏氨酸侧链（替代酪氨酸）可能具有催化作用。
