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Configuration of active site segments in lytic polysaccharide monooxygenases steers oxidative xyloglucan degradation.
Biotechnology for Biofuels ( IF 6.3 ) Pub Date : 2020-05-29 , DOI: 10.1186/s13068-020-01731-x
Peicheng Sun 1 , Christophe V F P Laurent 2, 3 , Stefan Scheiblbrandner 2 , Matthias Frommhagen 1 , Dimitrios Kouzounis 1 , Mark G Sanders 1 , Willem J H van Berkel 1 , Roland Ludwig 2 , Mirjam A Kabel 1
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Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that oxidatively cleave plant cell wall polysaccharides. LPMOs classified as fungal Auxiliary Activities family 9 (AA9) have been mainly studied for their activity towards cellulose; however, various members of this AA9 family have been also shown to oxidatively cleave hemicelluloses, in particularly xyloglucan (XG). So far, it has not been studied in detail how various AA9 LPMOs act in XG degradation, and in particular, how the mode-of-action relates to the structural configuration of these LPMOs. Two Neurospora crassa (Nc) LPMOs were found to represent different mode-of-action towards XG. Interestingly, the configuration of active site segments of these LPMOs differed as well, with a shorter Segment 1 (−Seg1) and a longer Segment 2 (+Seg2) present in NcLPMO9C and the opposite for NcLPMO9M (+Seg1−Seg2). We confirmed that NcLPMO9C cleaved the non-reducing end of unbranched glucosyl residues within XG via the oxidation of the C4-carbon. In contrast, we found that the oxidative cleavage of the XG backbone by NcLPMO9M occurred next to both unbranched and substituted glucosyl residues. The latter are decorated with xylosyl, xylosyl–galactosyl and xylosyl–galactosyl–fucosyl units. The relationship between active site segments and the mode-of-action of these NcLPMOs was rationalized by a structure-based phylogenetic analysis of fungal AA9 LPMOs. LPMOs with a −Seg1+Seg2 configuration clustered together and appear to have a similar XG substitution-intolerant cleavage pattern. LPMOs with the +Seg1−Seg2 configuration also clustered together and are reported to display a XG substitution-tolerant cleavage pattern. A third cluster contained LPMOs with a −Seg1−Seg2 configuration and no oxidative XG activity. The detailed characterization of XG degradation products released by LPMOs reveal a correlation between the configuration of active site segments and mode-of-action of LPMOs. In particular, oxidative XG-active LPMOs, which are tolerant and intolerant to XG substitutions are structurally and phylogenetically distinguished from XG-inactive LPMOs. This study contributes to a better understanding of the structure–function relationship of AA9 LPMOs.

中文翻译:

裂解多糖单加氧酶中活性位点片段的配置引导氧化木葡聚糖降解。

裂解多糖单加氧酶 (LPMO) 是一种强大的酶,可氧化切割植物细胞壁多糖。被归类为真菌辅助活性家族 9 (AA9) 的 LPMO 主要因其对纤维素的活性而被研究;然而,这个 AA9 家族的各种成员也被证明可以氧化裂解半纤维素,特别是木葡聚糖 (XG)。到目前为止,尚未详细研究各种 AA9 LPMO 如何在 XG 降解中起作用,特别是作用方式与这些 LPMO 的结构配置有何关系。发现两种粗糙脉孢菌 (Nc) LPMO 代表了对 XG 的不同作用方式。有趣的是,这些 LPMO 的活动站点段的配置也不同,NcLPMO9C 中存在较短的第 1 段 (-Seg1) 和较长的第 2 段 (+Seg2),而 NcLPMO9M (+Seg1-Seg2) 则相反。我们证实 NcLPMO9C 通过 C4 碳的氧化切割 XG 内未支化的葡糖基残基的非还原端。相反,我们发现 NcLPMO9M 对 XG 主链的氧化切割发生在未支化和取代的葡糖基残基旁边。后者用木糖基、木糖基-半乳糖基和木糖基-半乳糖基-岩藻糖基单元装饰。通过对真菌 AA9 LPMO 进行基于结构的系统发育分析,使活性位点片段与这些 NcLPMO 的作用方式之间的关系合理化。具有-Seg1+Seg2 配置的LPMO 聚集在一起,似乎具有类似的XG 替代不耐受切割模式。具有 +Seg1-Seg2 配置的 LPMO 也聚集在一起,据报道显示出 XG 替代耐受切割模式。第三个集群包含具有-Seg1-Seg2 配置且没有氧化 XG 活性的 LPMO。LPMO 释放的 XG 降解产物的详细表征揭示了活性位点段的配置与 LPMO 的作用方式之间的相关性。特别是,对 XG 取代耐受和不耐受的氧化性 XG 活性 LPMO 在结构和系统发育上与 XG 非活性 LPMO 不同。本研究有助于更好地理解 AA9 LPMO 的结构-功能关系。LPMO 释放的 XG 降解产物的详细表征揭示了活性位点段的配置与 LPMO 的作用方式之间的相关性。特别是,对 XG 取代耐受和不耐受的氧化性 XG 活性 LPMO 在结构和系统发育上与 XG 非活性 LPMO 不同。本研究有助于更好地理解 AA9 LPMO 的结构-功能关系。LPMO 释放的 XG 降解产物的详细表征揭示了活性位点段的配置与 LPMO 的作用方式之间的相关性。特别是,对 XG 取代耐受和不耐受的氧化性 XG 活性 LPMO 在结构和系统发育上与 XG 非活性 LPMO 不同。本研究有助于更好地理解 AA9 LPMO 的结构-功能关系。
更新日期:2020-05-29
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