Cryo-electron microscopy structure of the di-domain core of Mycobacterium tuberculosis polyketide synthase 13, essential for mycobacterial mycolic acid synthesis.

IF 2.6 4区 生物学 Q3 MICROBIOLOGY
Hannah E Johnston, Sarah M Batt, Alistair K Brown, Christos G Savva, Gurdyal S Besra, Klaus Fütterer
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Abstract

Mycobacteria are known for their complex cell wall, which comprises layers of peptidoglycan, polysaccharides and unusual fatty acids known as mycolic acids that form their unique outer membrane. Polyketide synthase 13 (Pks13) of Mycobacterium tuberculosis, the bacterial organism causing tuberculosis, catalyses the last step of mycolic acid synthesis prior to export to and assembly in the cell wall. Due to its essentiality, Pks13 is a target for several novel anti-tubercular inhibitors, but its 3D structure and catalytic reaction mechanism remain to be fully elucidated. Here, we report the molecular structure of the catalytic core domains of M. tuberculosis Pks13 (Mt-Pks13), determined by transmission cryo-electron microscopy (cryoEM) to a resolution of 3.4 Å. We observed a homodimeric assembly comprising the ketoacyl synthase (KS) domain at the centre, mediating dimerization, and the acyltransferase (AT) domains protruding in opposite directions from the central KS domain dimer. In addition to the KS-AT di-domains, the cryoEM map includes features not covered by the di-domain structural model that we predicted to contain a dimeric domain similar to dehydratases, yet likely lacking catalytic function. Analytical ultracentrifugation data indicate a pH-dependent equilibrium between monomeric and dimeric assembly states, while comparison with the previously determined structures of M. smegmatis Pks13 indicates architectural flexibility. Combining the experimentally determined structure with modelling in AlphaFold2 suggests a structural scaffold with a relatively stable dimeric core, which combines with considerable conformational flexibility to facilitate the successive steps of the Claisen-type condensation reaction catalysed by Pks13.

结核分枝杆菌多酮合成酶 13 的二域核心的冷冻电子显微镜结构,该酶对分枝杆菌霉菌酸的合成至关重要。
分枝杆菌因其复杂的细胞壁而闻名,细胞壁由多层肽聚糖、多糖和被称为霉菌酸的不常见脂肪酸组成,形成了独特的外膜。结核分枝杆菌(引起结核病的细菌)的多酮合成酶 13(Pks13)催化霉酚酸合成的最后一步,然后将其输出并组装到细胞壁中。由于其重要作用,Pks13 是几种新型抗结核抑制剂的靶标,但其三维结构和催化反应机制仍有待全面阐明。在此,我们报告了结核杆菌 Pks13(Mt-Pks13)催化核心结构域的分子结构,该结构通过透射低温电子显微镜(cryoEM)测定,分辨率为 3.4 Å。我们观察到一种同源二聚体,由位于中心的酮酰合成酶(KS)结构域和从中心 KS 结构域二聚体向相反方向突出的酰基转移酶(AT)结构域组成。除了 KS-AT 二结构域外,低温电子显微镜图还包括二结构域结构模型未涵盖的特征,我们预测这些特征包含一个类似于脱水酶的二聚结构域,但很可能缺乏催化功能。分析超速离心数据表明,单体和二聚体组装状态之间的平衡取决于 pH 值,而与之前确定的 M. smegmatis Pks13 结构的比较则表明了其结构的灵活性。将实验测定的结构与 AlphaFold2 中的建模相结合,表明该结构支架具有一个相对稳定的二聚体核心,它与相当大的构象灵活性相结合,促进了 Pks13 催化的克莱森型缩合反应的连续步骤。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Microbiology-Sgm
Microbiology-Sgm 生物-微生物学
CiteScore
4.60
自引率
7.10%
发文量
132
审稿时长
3.0 months
期刊介绍: We publish high-quality original research on bacteria, fungi, protists, archaea, algae, parasites and other microscopic life forms. Topics include but are not limited to: Antimicrobials and antimicrobial resistance Bacteriology and parasitology Biochemistry and biophysics Biofilms and biological systems Biotechnology and bioremediation Cell biology and signalling Chemical biology Cross-disciplinary work Ecology and environmental microbiology Food microbiology Genetics Host–microbe interactions Microbial methods and techniques Microscopy and imaging Omics, including genomics, proteomics and metabolomics Physiology and metabolism Systems biology and synthetic biology The microbiome.
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