King Jerry D, Harmer Nicholas J, Preston Andrew, Palmer Colin M, Rejzek Martin, Field Robert A, Blundell Tom L, Maskell Duncan J
Department of Veterinary Medicine, Madingley Road, University of Cambridge, Cambridge CB3 0ES, UK.
J Mol Biol. 2007 Nov 30;374(3):749-63. doi: 10.1016/j.jmb.2007.09.055. Epub 2007 Sep 26.
The pathogenic bacteria Bordetella parapertussis and Bordetella bronchiseptica express a lipopolysaccharide O antigen containing a polymer of 2,3-diacetamido-2,3-dideoxy-l-galacturonic acid. The O-antigen cluster contains three neighbouring genes that encode proteins belonging to the short-chain dehydrogenase/reductase (SDR) family, wbmF, wbmG and wbmH, and we aimed to elucidate their individual functions. Mutation and complementation implicate each gene in O-antigen expression but, as their putative sugar nucleotide substrates are not currently available, biochemical characterisation of WbmF, WbmG and WbmH is impractical at the present time. SDR family members catalyse a wide range of chemical reactions including oxidation, reduction and epimerisation. Because they typically share low sequence conservation, however, catalytic function cannot be predicted from sequence analysis alone. In this context, structural characterisation of the native proteins, co-crystals and small-molecule soaks enables differentiation of the functions of WbmF, WbmG and WbmH. These proteins exhibit typical SDR architecture and coordinate NAD. In the substrate-binding domain, all three enzymes bind uridyl nucleotides. WbmG contains a typical SDR catalytic TYK triad, which is required for oxidoreductase function, but the active site is devoid of additional acid-base functionality. Similarly, WbmH possesses a TYK triad, but an otherwise feature-poor active site. Consequently, 3,5-epimerase function can probably be ruled out for these enzymes. The WbmF active site contains conserved 3,5-epimerase features, namely, a positionally conserved cysteine (Cys133) and basic side chain (His90 or Asn213), but lacks the serine/threonine component of the SDR triad and therefore may not act as an oxidoreductase. The data suggest a pathway for synthesis of the O-antigen precursor UDP-2,3-diacetamido-2,3-dideoxy-l-galacturonic acid and illustrate the usefulness of structural data in predicting protein function.
副百日咳博德特氏菌和支气管败血博德特氏菌这两种病原菌表达一种脂多糖O抗原，该抗原含有由2,3 - 二乙酰氨基 - 2,3 - 二脱氧 - L - 半乳糖醛酸组成的聚合物。O抗原基因簇包含三个相邻基因，分别编码属于短链脱氢酶/还原酶（SDR）家族的蛋白质wbmF、wbmG和wbmH，我们旨在阐明它们各自的功能。突变和互补实验表明每个基因都参与O抗原的表达，但是由于目前尚未获得它们假定的糖核苷酸底物，因此目前对WbmF、WbmG和WbmH进行生化特性分析是不切实际的。SDR家族成员催化多种化学反应，包括氧化、还原和差向异构化。然而，由于它们通常序列保守性较低，仅通过序列分析无法预测催化功能。在这种情况下，对天然蛋白质、共晶体和小分子浸泡物进行结构表征能够区分WbmF、WbmG和WbmH的功能。这些蛋白质呈现典型的SDR结构并结合NAD。在底物结合结构域中，所有三种酶都结合尿苷核苷酸。WbmG含有典型的SDR催化三联体TYK，这是氧化还原酶功能所必需的，但活性位点缺乏额外的酸碱功能基团。同样，WbmH也拥有一个TYK三联体，但活性位点其他特征较少。因此，这些酶可能不具备3,5 - 差向异构酶功能。WbmF活性位点包含保守的3,5 - 差向异构酶特征，即位置保守的半胱氨酸（Cys133）和碱性侧链（His90或Asn213），但缺乏SDR三联体的丝氨酸/苏氨酸成分，因此可能不作为氧化还原酶起作用。这些数据表明了O抗原前体UDP - 2,3 - 二乙酰氨基 - 2,3 - 二脱氧 - L - 半乳糖醛酸的合成途径，并说明了结构数据在预测蛋白质功能方面的有用性。
