Department of Microbiology & Molecular Genetics, University of Texas Health Science Center, Houston, Texas, USA.
Department of Microbiology & Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
J Bacteriol. 2018 Apr 9;200(9). doi: 10.1128/JB.00783-17. Print 2018 May 1.
The actinobacterium has been implicated in nucleation of oral microbial consortia leading to biofilm formation. Due to the lack of genetic tools, little is known about basic cellular processes, including protein secretion and folding, in this organism. We report here a survey of the genome, which encodes a large number of exported proteins containing paired cysteine residues, and identified an oxidoreductase that is highly homologous to the thiol-disulfide oxidoreductase MdbA (MdbA). Crystallization studies uncovered that the 1.2-Å resolution structure of MdbA (MdbA) possesses two conserved features found in actinobacterial MdbA enzymes, a thioredoxin-like fold and an extended α-helical domain. By reconstituting the disulfide bond-forming machine , we demonstrated that MdbA catalyzes disulfide bond formation within the actinobacterial pilin FimA. A new gene deletion method supported that is essential in Remarkably, heterologous expression of MdbA in the Δ mutant rescued its known defects in cell growth and morphology, toxin production, and pilus assembly, and this thiol-disulfide oxidoreductase activity required the catalytic motif CXXC. Altogether, the results suggest that MdbA is a major thiol-disulfide oxidoreductase, which likely mediates posttranslocational protein folding in by a mechanism that is conserved in The actinobacterium has been implicated in the development of oral biofilms or dental plaque; however, little is known about the basic cellular processes in this organism. We report here a high-resolution structure of a oxidoreductase that is highly homologous to the thiol-disulfide oxidoreductase MdbA. By biochemical analysis, we demonstrated that MdbA catalyzes disulfide bond formation Furthermore, a new gene deletion method revealed that deletion of is lethal in Remarkably, MdbA can replace MdbA to maintain normal cell growth and morphology, toxin production, and pilus assembly. Overall, our studies support the hypothesis that utilizes MdbA as a major oxidoreductase to catalyze oxidative protein folding.
该放线菌被牵连到口腔微生物群落的形成导致生物膜形成的起始。由于缺乏遗传工具，人们对这个生物体的基本细胞过程，包括蛋白质分泌和折叠，知之甚少。我们在这里报告了对该基因组的调查，该基因组编码了大量含有配对半胱氨酸残基的分泌蛋白，并鉴定出一种氧化还原酶，该酶与硫氧还蛋白样折叠和延伸的α-螺旋结构域高度同源。晶体结构研究揭示了 1.2 埃分辨率的结构具有在放线菌 MdbA 酶中发现的两个保守特征，一种硫氧还蛋白样折叠和一个延伸的α-螺旋结构域。通过重新构建二硫键形成机器，我们证明 MdbA 催化了 actinobacterial pilin FimA 中的二硫键形成。一种新的基因缺失方法表明在 中是必需的。值得注意的是，在 中异源表达 MdbA 拯救了其已知的细胞生长和形态、毒素产生和菌毛组装缺陷，并且这种硫氧还蛋白-二硫键氧化还原酶活性需要催化基序 CXXC。总的来说，这些结果表明 MdbA 是一种主要的硫氧还蛋白-二硫键氧化还原酶，它可能通过一种在 中保守的机制来介导蛋白质的翻译后折叠。该放线菌与口腔生物膜或牙菌斑的形成有关；然而，人们对这个生物体的基本细胞过程知之甚少。我们在这里报告了一种与 硫氧还蛋白-二硫键氧化还原酶 MdbA 高度同源的 氧化还原酶的高分辨率结构。通过生化分析，我们证明了 MdbA 催化二硫键形成。此外，一种新的基因缺失方法表明，在 中缺失 是致命的。值得注意的是，MdbA 可以取代 MdbA 来维持正常的细胞生长和形态、毒素产生和菌毛组装。总的来说，我们的研究支持了这样一种假设，即 利用 MdbA 作为一种主要的氧化还原酶来催化氧化蛋白质折叠。
