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

探索糖苷水解酶多聚体家族的结构功能景观

基本信息

批准号：
BB/X016749/1
负责人：
Elisabeth Lowe
金额：
$ 59万
依托单位：
Newcastle University
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2023
资助国家：
英国
起止时间：
2023 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FX016749%2F1
关键词：
Exploring structure function landscape multimeric

项目摘要

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.

分枝杆菌如结核分枝杆菌具有复杂的细胞壁，包括含有一种不寻常糖的聚糖和脂聚糖；D-arabinose。这种糖很少在分枝杆菌及其近亲之外发现。我们已经发现了一个新的苷水解酶家族（GH172s），它能够降解这种细胞壁。这些酶中的一些来自肠道细菌，它们精通人类远端肠道中复杂聚糖的降解，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。一些家族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。含有聚糖的d -阿拉伯糖对分枝杆菌的生存至关重要，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d -阿拉伯糖靶向致病性分枝杆菌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。表征肠道细菌d -阿拉伯糖降解酶的工具箱将用于分枝杆菌细胞壁研究，诊断和新疗法的开发。不同寻常的是，这个酶家族是多聚的，活性位点形成在单体之间的界面上。我们有结构数据显示2-，3- 0.6 -和12-mers的酶组合，但我们还不了解多聚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，我们必须探究它们的精确特异性和活性。在本提案中，我们的目标是表征GH172家族的不同成员，从共生肠道微生物和病原体。我们将剖析序列、特异性和多聚合之间的关系，为未表征的GH172成员提供预测基序。我们的主要目标是：1)表征来自共生体和病原体的各种GH172酶；2)了解这些酶是如何多聚的以及它是如何影响活性的；3)使用低温电子显微镜发现这些多聚酶是如何进入分枝杆菌细胞壁的异质支链聚糖的。我们将有一套具有明确活性的酶来对抗含有d -阿拉伯糖的聚糖，这将对糖生物学家和分枝杆菌学家有广泛的用途。这些酶存在于鸟分枝杆菌复合体（MAC）的成员中，这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群中。MAC感染治疗困难，复发率高。在世界范围内，这些非结核分枝杆菌感染的发病率正在增加。在牲畜约翰氏病的病原体鸟分枝杆菌副结核杆菌中也发现了GH172酶。这是全世界牛群面临的一个主要问题。导致消耗性疾病、产奶量减少、体重下降甚至死亡。了解GH172细胞壁靶向酶的作用可能有助于开发针对这些感染的新疗法或诊断方法。