Exploring the structure-function landscape of a multimeric family of glycoside hydrolases

分枝杆菌，如结核分枝杆菌，有一个复杂的细胞壁，包括含有一种不寻常的糖--D-阿拉伯糖的多糖和脂多糖。这种糖在分枝杆菌及其近亲之外很少发现。我们已经确定了一个新的糖苷水解酶家族(GH172s)，它能够降解这个细胞壁。其中几种酶来自肠道细菌，这种细菌擅长降解人类末端肠道中的复杂多糖，但我们也在人类和动物分枝杆菌病原体中发现了这些酶的同源物。一些家庭成员也能够瞄准低聚果糖，这是人类饮食中的一种常见成分。含有D-阿拉伯糖的多糖对分枝杆菌的生存是必不可少的，事实上，它们的合成机械是一线抗结核药物乙胺丁醇的靶标。了解致病分枝杆菌D-阿拉伯聚糖酶的特异性，可以提供未来的药物靶点，并改变我们对分枝杆菌如何重塑或回收细胞壁的理解。肠道细菌D-阿拉伯聚糖降解酶的工具箱将用于分枝杆菌细胞壁的研究、诊断和新疗法的开发。不同寻常的是，这一酶家族是多聚体的，活性部位形成在单体之间的界面上。我们有结构数据显示2-，3-.6-和12-MERS的酶组装，但我们还不知道多聚体对酶功能或特异性的贡献。为了充分了解这些酶的生物学作用，我们必须询问它们准确的特异性和活性。在这项建议中，我们的目标是从共生肠道微生物和病原体中鉴定GH172家族的不同成员。我们将剖析序列、特异性和多聚体之间的关系，为未具体化的GH172成员提供预测基序。我们的主要目标是：1)从共生体和病原体表征不同的GH172酶2)了解这些酶是如何多聚化的以及它是如何影响活性的3)使用低温电子显微镜来发现这些多聚体酶是如何访问分枝杆菌细胞壁的异质支链多糖的结果除了拓宽我们对一个不寻常的酶家族的理解外，我们还将拥有一套对含有D-阿拉伯糖的糖链具有明确活性的酶，这对糖生物学家和分枝细菌学家将是广泛的有用的。这些酶是在鸟分枝杆菌复合体(MAC)的成员中发现的，MAC是一组导致许多肺部感染的物种，特别是在免疫功能低下的人中。MAC感染治疗困难，复发率高。这些非结核分枝杆菌感染的发病率在全世界范围内都在增加。在禽类副结核杆菌中也发现了一种GH172酶，这是家畜约翰氏病的病原体。这对牛群来说是一个重大的世界性问题。导致疾病消瘦、产奶量减少、体重减轻甚至死亡。了解GH172细胞壁靶向酶的作用可能会导致开发针对这些感染的新疗法或诊断方法。

Mycobacteria such as M. tuberculosis have a complex cell wall, including glycans and lipoglycans which contain an unusual sugar; D-arabinose. This sugar is rarely found outside mycobacteria and their relatives. We have identified a new family of glycoside hydrolase enzymes (GH172s) which are able to degrade this cell wall. Several of these enzymes are from gut bacteria which are proficient in degradation of complex glycans in the human distal gut, but we have also identified homologs of these enzymes in human and animal mycobacterial pathogens. Some family members are also able to target fructo-oligosaccharides, a common component of the human diet. D-arabinose containing glycans are essential to mycobacterial viability, indeed, their synthetic machinery is the target of a front-line anti-tuberculosis drug, Ethambutol. Understanding the specificity of D-arabinan targeting enzymes from pathogenic mycobacteria could provide future drug targets, and transform our understanding of how mycobacteria may remodel or recycle their cell wall. A toolbox of characterised gut bacterial D-arabinan degrading enzymes will be of use to mycobacterial cell wall research, diagnostics, and development of new therapeutics.Unusually, this family of enzymes is multimeric, with the active site formed at the interface between monomers. We have structural data showing enzyme assemblies of 2-,3-.6- and 12-mers, but we do not yet understand the contribution of multimerisation to enzyme function or specificity. To fully understand the biological role of these enzymes, we must interrogate their precise specificity and activity.In this proposal we aim to characterise diverse members of the GH172 family, from both commensal gut microbes and pathogens. We will dissect the relationship between sequence, specificity and multimerisation, to provide predictive motifs for uncharacterised GH172 members. Our main aims are:1) characterise diverse GH172 enzymes from commensals and pathogens2) understand how these enzymes multimerise and how it affects activity3) use cryo electron microscopy to discover how these multimeric enzymes access the heterogeneous, branched glycans of the mycobacterial cell wallOutcomesIn addition to broadening our understanding of an unusual family of enzymes, we will have a suite of enzymes with defined activities against D-arabinose containing glycans that will be of broad utility to glycobiologists and mycobacteriologists. These enzymes are found in members of the Mycobacterium avium complex (MAC), a group of species that are responsible for many pulmonary infections, particularly in immunocompromised people. Treatment of MAC infections is difficult, and reoccurrence is high. The incidence of these non-tuberculous mycobacterial infections is increasing worldwide. A GH172 enzyme is also found in M. avium paratuberculosis, the causative agent of Johne's disease in livestock. This is a major worldwide problem for cattle herds. leading to wasting disease, reduced milk yields, weight loss and even death. Understanding the role of GH172 cell-wall targeting enzymes could lead to the development of new therapeutics or diagnostics for these infections.