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

分枝杆菌如M.结核杆菌具有复杂的细胞壁，包括聚糖和脂聚糖，其中含有不寻常的糖; D-阿拉伯糖。这种糖在分枝杆菌及其亲属之外很少发现。我们已经确定了一个新的糖苷水解酶家族（GH 172），能够降解这种细胞壁。这些酶中有几种来自肠道细菌，这些细菌擅长降解人类远端肠道中的复合聚糖，但我们也在人类和动物分枝杆菌病原体中鉴定了这些酶的同源物。一些家庭成员也能够针对低聚果糖，这是人类饮食的一种常见成分。含有D-阿拉伯糖的聚糖对分枝杆菌的生存能力至关重要，事实上，它们的合成机制是一线抗结核药物乙胺丁醇的目标。了解致病性分枝杆菌D-阿拉伯聚糖靶向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其细胞壁的理解。一个工具箱的特点肠道细菌D-阿拉伯聚糖降解酶将用于分枝杆菌细胞壁的研究，诊断，和开发新的therapeutics.Unusually，这个家庭的酶是多聚体，与活性位点形成在单体之间的界面。我们有显示2-、3-、6-和12-聚体的酶组装体的结构数据，但我们还不了解多聚化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，我们必须询问它们的精确特异性和活性。在这项提议中，我们的目标是从肠道微生物和病原体中分离GH 172家族的不同成员。我们将剖析序列，特异性和多聚化之间的关系，为未表征的GH 172成员提供预测基序。我们的主要目的是：1）从细菌和病原体中获得多种多样的GH 172酶2）了解这些酶如何多聚化以及它如何影响活性3）使用冷冻电子显微镜来发现这些多聚酶如何进入分枝杆菌细胞壁的异质性分支聚糖3）结果除了扩大我们对一个不寻常的酶家族的理解外，我们将有一套对含D-阿拉伯糖聚糖具有确定活性的酶，这将对糖生物学家和分枝杆菌学家具有广泛的实用性。这些酶存在于鸟分枝杆菌复合体（MAC）的成员中，这是一组负责许多肺部感染的物种，特别是在免疫功能低下的人群中。MAC感染的治疗很困难，复发率很高。这些非结核分枝杆菌感染的发病率在世界范围内不断增加。在M.禽副结核病，家畜约翰氏病的病原体。这是一个严重的世界性问题的牛群。导致消耗性疾病、产奶量减少、体重减轻甚至死亡。了解GH 172细胞壁靶向酶的作用可能会导致这些感染的新疗法或诊断的发展。

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.