In vitro and in vivo characterization of three Cellvibrio japonicus glycoside hydrolase family 5 members reveals potent xyloglucan backbone-cleaving functions.
In vitro and in vivo characterization of three Cellvibrio japonicus glycoside hydrolase family 5 members reveals potent xyloglucan backbone-cleaving functions.
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DOI：
10.1186/s13068-018-1039-6
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发表时间：
2018
影响因子：
6.3
通讯作者：
Brumer H
中科院分区：
文献类型：
--
作者：
Attia MA;Nelson CE;Offen WA;Jain N;Davies GJ;Gardner JG;Brumer H
木葡聚糖（XyG）是植物细胞壁中普遍存在的基本多糖。由于其结构的复杂性，XyG需要主链裂解和侧链脱支酶的组合来完全解构成其组分单糖。土壤嗜盐菌纤维弧菌（Cellvibrio）已经成为研究生物质糖化的遗传上易于处理的模型系统，部分原因是其利用广泛的植物多糖用于生长的固有能力。而C.尽管已经对其进行了功能表征，但所需的主链裂解内切木葡聚糖酶尚未得到解决。结合生物信息学和转录组学的分析表明，三个糖苷水解酶家族5亚家族4（GH5_4）成员，具有不同的模块组织，作为潜在的关键内切木葡聚糖酶在C。- 是的所有三种重组蛋白的GH5_4模块的详细生物化学和酶表征证实了XyG多糖相对于一组其他细胞壁聚糖（包括混合键β-葡聚糖和纤维素）的特别高的特异性。此外，产物分析表明，所有三种酶产生的XyG寡糖需要后续糖化已知的外切糖苷酶。GH 5D是在XyG上生长期间特异性和高度上调的唯一GH5_4成员，其以游离、产物复合物和活性位点亲和标记的形式的晶体学分析揭示了这些GH5_4酶之间的精致XyG特异性的分子基础。引人注目的是，对所有三个GH5_4成员和一个先前生物化学表征的GH 74成员的详尽的反向遗传分析未能揭示生长缺陷，从而表明在该群组的成员之间以及通过C. - 是的我们基于系统的分析表明，不同的底物传感（GH 74，GH 5E，GH5 F）和攻击安装（GH 5D）功能的内切木葡聚糖酶的特点。本研究从分子系统的角度对木葡聚糖利用系统的糖化途径进行了深入的研究。- 是的三种不同的GH 5_4内切木葡聚糖酶的详细结构-功能表征将为未来跨物种的生物信息学预测提供信息，并提供具有明确特异性的新CAZymes，可用于工业和其他生物技术应用。本文的在线版本（10.1186/s13068-018-1039-6）包含补充材料，可供授权用户使用。
Xyloglucan (XyG) is a ubiquitous and fundamental polysaccharide of plant cell walls. Due to its structural complexity, XyG requires a combination of backbone-cleaving and sidechain-debranching enzymes for complete deconstruction into its component monosaccharides. The soil saprophyte Cellvibrio japonicus has emerged as a genetically tractable model system to study biomass saccharification, in part due to its innate capacity to utilize a wide range of plant polysaccharides for growth. Whereas the downstream debranching enzymes of the xyloglucan utilization system of C. japonicus have been functionally characterized, the requisite backbone-cleaving endo-xyloglucanases were unresolved. Combined bioinformatic and transcriptomic analyses implicated three glycoside hydrolase family 5 subfamily 4 (GH5_4) members, with distinct modular organization, as potential keystone endo-xyloglucanases in C. japonicus. Detailed biochemical and enzymatic characterization of the GH5_4 modules of all three recombinant proteins confirmed particularly high specificities for the XyG polysaccharide versus a panel of other cell wall glycans, including mixed-linkage beta-glucan and cellulose. Moreover, product analysis demonstrated that all three enzymes generated XyG oligosaccharides required for subsequent saccharification by known exo-glycosidases. Crystallographic analysis of GH5D, which was the only GH5_4 member specifically and highly upregulated during growth on XyG, in free, product-complex, and active-site affinity-labelled forms revealed the molecular basis for the exquisite XyG specificity among these GH5_4 enzymes. Strikingly, exhaustive reverse-genetic analysis of all three GH5_4 members and a previously biochemically characterized GH74 member failed to reveal a growth defect, thereby indicating functional compensation in vivo, both among members of this cohort and by other, yet unidentified, xyloglucanases in C. japonicus. Our systems-based analysis indicates distinct substrate-sensing (GH74, GH5E, GH5F) and attack-mounting (GH5D) functions for the endo-xyloglucanases characterized here. Through a multi-faceted, molecular systems-based approach, this study provides a new insight into the saccharification pathway of xyloglucan utilization system of C. japonicus. The detailed structural–functional characterization of three distinct GH5_4 endo-xyloglucanases will inform future bioinformatic predictions across species, and provides new CAZymes with defined specificity that may be harnessed in industrial and other biotechnological applications. The online version of this article (10.1186/s13068-018-1039-6) contains supplementary material, which is available to authorized users.
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影响因子：
3.5
作者：
BARRAS, F;BORTOLIGERMAN, I;HENRISSAT, B
通讯作者：
HENRISSAT, B
影响因子：
3.4
作者：
Aspeborg H;Coutinho PM;Wang Y;Brumer H 3rd;Henrissat B
通讯作者：
Henrissat B
影响因子：
5.4
作者：
Attia, Mohamed;Stepper, Judith;Brumer, Harry
通讯作者：
Brumer, Harry
DOI：
10.1107/s0907444909042073
发表时间：
2010-01
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Chen VB;Arendall WB 3rd;Headd JJ;Keedy DA;Immormino RM;Kapral GJ;Murray LW;Richardson JS;Richardson DC
通讯作者：
Richardson DC
影响因子：
6.8
作者：
Attia, Mohamed A.;Brumer, Harry
通讯作者：
Brumer, Harry