A Disulfide Oxidoreductase (CHU_1165) Is Essential for Cellulose Degradation by Affecting Outer Membrane Proteins in Cytophaga hutchinsonii
A Disulfide Oxidoreductase (CHU_1165) Is Essential for Cellulose Degradation by Affecting Outer Membrane Proteins in Cytophaga hutchinsonii
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二硫键氧化还原酶 (CHU_1165) 通过影响哈钦森噬细胞菌的外膜蛋白而对纤维素降解至关重要
DOI：
10.1128/aem.02789-19
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发表时间：
2020-02
影响因子：
4.4
通讯作者：
Lu Xuemei
中科院分区：
文献类型：
--
作者：
Zhao Dong;Wang Ying;Wang Sen;Zhang Weican;Qi Qingsheng;Lu Xuemei
胡氏嗜纤维菌能以接触依赖的方式快速消化纤维素，外膜蛋白可能在其中起重要作用。在这项研究中，一个假设的蛋白质，CHU_1165，其特征是一个二硫键氧化还原酶，是必不可少的纤维素降解，通过影响许多外膜蛋白的纤维素结合能力，在C。哈钦森氏菌二硫氧化还原酶参与二硫键的形成。然而，我们的研究表明，许多减少的外膜蛋白是纤维素降解所必需的，不含或含有一个半胱氨酸，半胱氨酸的突变并不影响其功能，表明CHU_1165并不促进这些蛋白质中二硫键的形成。它可能对这些外膜蛋白的功能具有间接或多效性作用。我们的研究为探索纤维素降解所必需的许多外膜蛋白的适当构象提供了方向，这对于探索C.哈钦森氏菌摘要胡氏噬纤维菌（Cytophagahutchinsonii）细胞能与不溶性纤维素表面结合并利用一种新的细胞接触依赖性机制降解纤维素，其中外膜蛋白可能起重要作用。在本研究中，一个基因位点chu_1165的缺失导致了C.哈钦森氏菌进一步的研究表明，Δ1165菌株的细胞不能与纤维素结合，许多能与纤维素结合的外膜蛋白的水平显著降低。CHU_1165的N-末端区域锚定到细胞质膜上，具有5个预测的跨膜螺旋，C-末端区域预测延伸到周质，并具有类似的硫氧还蛋白（Trx）折叠，其中含有二硫键氧化还原酶中保守的Cys-X-X-Cys基序。含Cys-X-X-Cys基序的重组CHU_1165His在体外能够还原胰岛素的二硫键。定点突变结果表明，CHU_1165的Cys-X-X-Cys基序和第106、108位残基的半胱氨酸对CHU_1165的功能是必需的。Western blotting结果表明，CHU_1165在体内处于氧化状态，提示其可能作为氧化酶催化二硫键的形成。然而，许多减少的纤维素降解所必需的外膜蛋白不含或含有一个半胱氨酸，并且这些蛋白中的半胱氨酸突变不影响纤维素降解，表明CHU_1165可能对这些外膜蛋白的功能具有间接或多效性作用。重要性Cytophaga hutchinsonii能够以接触依赖的方式快速消化纤维素，其中外膜蛋白可能起重要作用。在这项研究中，一个假设的蛋白质，CHU_1165，其特征是一个二硫键氧化还原酶，是必不可少的纤维素降解，通过影响许多外膜蛋白的纤维素结合能力，在C。哈钦森氏菌二硫氧化还原酶参与二硫键的形成。然而，我们的研究表明，许多减少的外膜蛋白是纤维素降解所必需的，不含或含有一个半胱氨酸，半胱氨酸的突变并不影响其功能，表明CHU_1165并不促进这些蛋白质中二硫键的形成。它可能对这些外膜蛋白的功能具有间接或多效性作用。我们的研究为探索纤维素降解所必需的许多外膜蛋白的适当构象提供了方向，这对于探索C.哈钦森氏菌
Cytophaga hutchinsonii can rapidly digest cellulose in a contact-dependent manner, in which the outer membrane proteins may play important roles. In this study, a hypothetical protein, CHU_1165, characterized as a disulfide oxidoreductase, is essential for cellulose degradation by affecting the cellulose binding ability of many outer membrane proteins in C. hutchinsonii. Disulfide oxidoreductases are involved in disulfide bond formation. However, our studies show that many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of cysteine did not affect their function, indicating that CHU_1165 did not facilitate the formation of a disulfide bond in these proteins. It may have an indirect or pleiotropic effect on the function of these outer membrane proteins. Our study provides an orientation for exploring the proteins that assist in the appropriate conformation of many outer membrane proteins essential for cellulose degradation, which is important for exploring the novel mechanism of cellulose degradation in C. hutchinsonii. ABSTRACT Cytophaga hutchinsonii cells can bind to the surface of insoluble cellulose and degrade it by utilizing a novel cell contact-dependent mechanism, in which the outer membrane proteins may play important roles. In this study, the deletion of a gene locus, chu_1165, which encodes a hypothetical protein with 32% identity with TlpB, a disulfide oxidoreductase in Flavobacterium psychrophilum, caused a complete cellulolytic defect in C. hutchinsonii. Further study showed that cells of the Δ1165 strain could not bind to cellulose, and the levels of many outer membrane proteins that can bind to cellulose were significantly decreased. The N-terminal region of CHU_1165 is anchored to the cytoplasmic membrane with five predicted transmembrane helices, and the C-terminal region is predicted to stretch to the periplasm and has a similar thioredoxin (Trx) fold containing a Cys-X-X-Cys motif that is conserved in disulfide oxidoreductases. Recombinant CHU_1165His containing the Cys-X-X-Cys motif was able to reduce the disulfide bonds of insulin in vitro. Site-directed mutation showed that the cysteines in the Cys-X-X-Cys motif and at residues 106 and 108 were indispensable for the function of CHU_1165. Western blotting showed that CHU_1165 was in an oxidized state in vivo, suggesting that it may act as an oxidase to catalyze disulfide bond formation. However, many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of the cysteine in these proteins did not affect cellulose degradation, indicating that CHU_1165 may have an indirect or pleiotropic effect on the function of these outer membrane proteins. IMPORTANCE Cytophaga hutchinsonii can rapidly digest cellulose in a contact-dependent manner, in which the outer membrane proteins may play important roles. In this study, a hypothetical protein, CHU_1165, characterized as a disulfide oxidoreductase, is essential for cellulose degradation by affecting the cellulose binding ability of many outer membrane proteins in C. hutchinsonii. Disulfide oxidoreductases are involved in disulfide bond formation. However, our studies show that many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of cysteine did not affect their function, indicating that CHU_1165 did not facilitate the formation of a disulfide bond in these proteins. It may have an indirect or pleiotropic effect on the function of these outer membrane proteins. Our study provides an orientation for exploring the proteins that assist in the appropriate conformation of many outer membrane proteins essential for cellulose degradation, which is important for exploring the novel mechanism of cellulose degradation in C. hutchinsonii.
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作者：
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影响因子：
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DOI：
10.1007/s10295-019-02212-3
发表时间：
2019-07
影响因子：
3.4
作者：
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通讯作者：
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