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Defining natural factors that stimulate and inhibit cellulose:xyloglucan hetero‐transglucosylation
The Plant Journal ( IF 6.2 ) Pub Date : 2020-12-13 , DOI: 10.1111/tpj.15131
Klaus Herburger 1 , Lenka Franková 1 , Martina Pičmanová 1 , Anzhou Xin 1 , Frank Meulewaeter 2 , Andrew Hudson 1 , Stephen C Fry 1
Affiliation  

Certain transglucanases can covalently graft cellulose and mixed‐linkage β‐glucan (MLG) as donor substrates onto xyloglucan as acceptor substrate and thus exhibit cellulose:xyloglucan endotransglucosylase (CXE) and MLG:xyloglucan endotransglucosylase (MXE) activities in vivo and in vitro. However, missing information on factors that stimulate or inhibit these hetero‐transglucosylation reactions limits our insight into their biological functions. To explore factors that influence hetero‐transglucosylation, we studied Equisetum fluviatile hetero‐trans‐β‐glucanase (EfHTG), which exhibits both CXE and MXE activity, exceeding its xyloglucan:xyloglucan homo‐transglucosylation (XET) activity. Enzyme assays employed radiolabelled and fluorescently labelled oligomeric acceptor substrates, and were conducted in vitro and in cell walls (in situ). With whole denatured Equisetum cell walls as donor substrate, exogenous EfHTG (extracted from Equisetum or produced in Pichia) exhibited all three activities (CXE, MXE, XET) in competition with each other. Acting on pure cellulose as donor substrate, the CXE action of Pichia‐produced EfHTG was up to approximately 300% increased by addition of methanol‐boiled Equisetum extracts; there was no similar effect when the same enzyme acted on soluble donors (MLG or xyloglucan). The methanol‐stable factor is proposed to be expansin‐like, a suggestion supported by observations of pH dependence. Screening numerous low‐molecular‐weight compounds for hetero‐transglucanase inhibition showed that cellobiose was highly effective, inhibiting the abundant endogenous CXE and MXE (but not XET) action in Equisetum internodes. Furthermore, cellobiose retarded Equisetum stem elongation, potentially owing to its effect on hetero‐transglucosylation reactions. This work provides insight and tools to further study the role of cellulose hetero‐transglucosylation in planta by identifying factors that govern this reaction.

中文翻译:

定义刺激和抑制纤维素的自然因素:木葡聚糖异葡糖基化

某些转葡聚糖酶可以将纤维素和混合键β-葡聚糖(MLG)作为供体底物共价接枝到作为受体底物的木葡聚糖上,从而在体内体外表现出纤维素:木葡聚糖内转葡糖基酶(CXE)和MLG:木葡聚糖内转葡糖基酶(MXE)活性。然而,缺少有关刺激或抑制这些异质转葡萄糖基化反应的因素的信息限制了我们对其生物学功能的了解。为了探索影响异质转葡糖基化的因素,我们研究了木贼属河流异质转β-葡聚糖酶(EfHTG),表现出 CXE 和 MXE 活性,超过其木葡聚糖:木葡聚糖同型转葡糖基化 (XET) 活性。酶测定采用放射性标记和荧光标记的寡聚受体底物,并在体外和细胞壁(原位)中进行。以整个变性木贼细胞壁作为供体底物,外源性Ef HTG(从木贼属中提取或在毕赤酵母中生产)表现出所有三种活性(CXE、MXE、XET),相互竞争。作用于纯纤维素作为供体底物,毕赤酵母产生的Ef HTG的 CXE 作用通过添加甲醇煮沸提高了约 300%木贼提取物;当相同的酶作用于可溶性供体(MLG 或木葡聚糖)时,没有类似的效果。甲醇稳定因子被认为是类似扩展蛋白的,这一建议得到了 pH 依赖性观察的支持。筛选多种低分子量化合物以抑制异质转葡聚糖酶表明,纤维二糖非常有效,可抑制木贼属植物节间丰富的内源性 CXE 和 MXE(但不是 XET)作用。此外,纤维二糖延缓了木贼茎的伸长,这可能是由于其对异质转葡萄糖基化反应的影响。这项工作提供了洞察力和工具,通过确定控制该反应的因素,进一步研究纤维素异质转葡糖基化在植物中的作用。
更新日期:2020-12-13
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