细菌酪氨酸假激酶EcpK在黄粘球菌胞外多糖生物合成中重要作用的鉴定。

IF 2.7 3区 生物学 Q3 MICROBIOLOGY
Luca Blöcher, Johannes Schwabe, Timo Glatter, Lotte Søgaard-Andersen
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引用次数: 0

摘要

细菌合成化学上多样的荚膜多糖和分泌多糖,这些多糖在许多生理过程中起作用,并在工业上得到广泛应用。在这些多糖普遍存在的Wzx/ wzy依赖的生物合成途径中,多糖共聚合酶(PCP)促进了周质中重复单元的聚合,并且在革兰氏阴性菌中,也促进了多糖跨外膜的转运。这些pcp属于PCP-2家族,是具有扩展质周结构域的完整内膜蛋白,其功能依赖于不同寡聚物状态之间的交替。寡聚状态是由同源细胞质细菌酪氨酸激酶(BYK)决定的,它是PCP的一部分或一个独立的蛋白质。有趣的是,已经描述了缺乏关键催化残基和/或磷酸化Tyr残基的byk样蛋白。在黄粘球菌中,胞外多糖(EPS)通过Wzx/ wzy依赖性EPS途径合成和输出,其中EpsV作为PCP。在这里,我们证实EpsV缺乏BYK结构域。利用系统基因组学、实验和计算结构生物学,我们发现EcpK对EPS生物合成很重要,并表明它在结构上类似于典型的byk,但缺乏催化和Tyr磷酸化的重要残基。通过蛋白质组学分析、双杂交分析和结构建模,我们证明了EcpK直接与EpsV相互作用。基于这些发现,我们认为EcpK是一种BY假激酶,并作为支架,通过直接的蛋白质-蛋白质相互作用,而不是通过Tyr磷酸化,促进EpsV的功能。EcpK和EpsV的同源物存在于其他细菌中,表明该机制广泛保守,并建立了磷酸化不依赖的PCP-2亚家族。细菌产生多种具有重要生物学功能的多糖。在革兰氏阴性菌分泌多糖和荚膜多糖生物合成的Wzx/ wzy依赖途径中,多糖共聚合酶(PCP)是促进重复单元聚合和多糖跨外膜转运的关键蛋白。PCP的功能取决于由相关细菌酪氨酸激酶(BYK)的磷酸化/去磷酸化周期决定的组装/拆卸周期。在这里,我们确定了BY假激酶EcpK在黄粘球菌的胞外多糖生物合成中是必不可少的。基于实验和计算结构生物学,我们认为EcpK是一种支架蛋白,通过独立于Tyr磷酸化/去磷酸化周期的结合/解结合周期指导伴侣PCP的组装/拆卸周期。我们认为这种新机制是广泛保守的。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Identification of EcpK, a bacterial tyrosine pseudokinase important for exopolysaccharide biosynthesis in Myxococcus xanthus.

Bacteria synthesize chemically diverse capsular and secreted polysaccharides that function in many physiological processes and are widely used in industrial applications. In the ubiquitous Wzx/Wzy-dependent biosynthetic pathways for these polysaccharides, the polysaccharide co-polymerase (PCP) facilitates the polymerization of repeat units in the periplasm, and in Gram-negative bacteria, also polysaccharide translocation across the outer membrane. These PCPs belong to the PCP-2 family, are integral inner membrane proteins with extended periplasmic domains, and functionally depend on alternating between different oligomeric states. The oligomeric state is determined by a cognate cytoplasmic bacterial tyrosine kinase (BYK), which is either part of the PCP or a stand-alone protein. Interestingly, BYK-like proteins, which lack key catalytic residues and/or the phosphorylated Tyr residues, have been described. In Myxococcus xanthus, the exopolysaccharide (EPS) is synthesized and exported via the Wzx/Wzy-dependent EPS pathway in which EpsV serves as the PCP. Here, we confirm that EpsV lacks the BYK domain. Using phylogenomics, experiments, and computational structural biology, we identify EcpK as important for EPS biosynthesis and show that it structurally resembles canonical BYKs but lacks residues important for catalysis and Tyr phosphorylation. Using proteomic analyses, two-hybrid assays, and structural modeling, we demonstrate that EcpK directly interacts with EpsV. Based on these findings, we suggest that EcpK is a BY pseudokinase and functions as a scaffold, which by direct protein-protein interactions, rather than by Tyr phosphorylation, facilitates EpsV function. EcpK and EpsV homologs are present in other bacteria, suggesting broad conservation of this mechanism and establishing a phosphorylation-independent PCP-2 subfamily.IMPORTANCEBacteria produce a variety of polysaccharides with important biological functions. In Wzx/Wzy-dependent pathways for the biosynthesis of secreted and capsular polysaccharides in Gram-negative bacteria, the polysaccharide co-polymerase (PCP) is a key protein that facilitates repeat unit polymerization and polysaccharide translocation across the outer membrane. PCP function depends on assembly/disassembly cycles that are determined by the phosphorylation/dephosphorylation cycles of an associated bacterial tyrosine kinase (BYK). Here, we identify the BY pseudokinase EcpK as essential for exopolysaccharide biosynthesis in Myxococcus xanthus. Based on experiments and computational structural biology, we suggest that EcpK is a scaffold protein, guiding the assembly/disassembly cycles of the partner PCP via binding/unbinding cycles independently of Tyr phosphorylation/dephosphorylation cycles. We suggest that this novel mechanism is broadly conserved.

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来源期刊
Journal of Bacteriology
Journal of Bacteriology 生物-微生物学
CiteScore
6.10
自引率
9.40%
发文量
324
审稿时长
1.3 months
期刊介绍: The Journal of Bacteriology (JB) publishes research articles that probe fundamental processes in bacteria, archaea and their viruses, and the molecular mechanisms by which they interact with each other and with their hosts and their environments.
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