Defining natural factors that stimulate and inhibit cellulose:xyloglucan hetero-transglucosylation.
Defining natural factors that stimulate and inhibit cellulose:xyloglucan hetero-transglucosylation.
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DOI：
10.1111/tpj.15131
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发表时间：
2021-03
期刊：
影响因子：
--
通讯作者：
Fry SC
中科院分区：
文献类型：
--
作者：
Herburger K;Franková L;Pičmanová M;Xin A;Meulewaeter F;Hudson A;Fry SC
某些转葡聚糖酶可以将作为供体底物的纤维素和混合键β-葡聚糖（MLG）共价接枝到作为受体底物的木葡聚糖上，因此在体内和体外表现出纤维素：木葡聚糖内切转葡糖基酶（CXE）和MLG：木葡聚糖内切转葡糖基酶（MXE）活性。然而，缺乏刺激或抑制这些异转葡萄糖基化反应的因子的信息限制了我们对其生物学功能的了解。为了探索影响杂转葡糖基化的因素，我们研究了木贼杂转β-葡聚糖酶（EfHTG），其表现出CXE和MXE活性，超过其木葡聚糖：木葡聚糖同转葡糖基化（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。以变性木贼细胞壁为供体底物，外源EfHTG（木贼提取物或毕赤酵母生产物）表现出CXE、MXE、XET三种活性，并相互竞争。作用于作为供体底物的纯纤维素时，通过添加甲醇煮沸的木贼提取物，毕赤酵母产生的EfHTG的CXE作用增加高达约300%;当相同的酶作用于可溶性供体（MLG或木葡聚糖）时，没有类似的作用。甲醇稳定因子被认为是膨胀素样的，这一建议得到了pH依赖性观察结果的支持。筛选许多低分子量化合物用于抑制异葡聚糖转移酶，表明纤维二糖非常有效，抑制木贼节间中丰富的内源性CXE和MXE（但不抑制XET）作用。此外，纤维二糖延迟木贼茎伸长，可能是由于其对异转葡萄糖基化反应的影响。这项工作提供了洞察力和工具，通过确定控制这种反应的因素，进一步研究纤维素异转葡萄糖基化在植物中的作用。HTG酶可以将纤维素分子的片段接枝到木葡聚糖（一种半纤维素）上，从而通过杂转糖基化来重构细胞壁。在天然壁中，两种内源性半纤维素与作为底物的纤维素（以及彼此）竞争。如果加入富含膨胀蛋白的制剂，HTG更容易选择纤维素作为底物。纤维二糖抑制了HTG转糖基化-这是探索HTG生物学功能的潜在工具。有趣的是，纤维二糖延缓木贼茎伸长，表明HTG在生长中的作用。
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. The enzyme HTG can graft segments of cellulose molecules onto xyloglucan (a hemicellulose), thereby re‐structuring the cell wall via hetero‐transglycosylation. In native walls, two endogenous hemicelluloses competed with cellulose (and with each other) as substrate. HTG more readily selected cellulose as substrate if an expansin‐enriched preparation was added. Hetero‐transglycosylation was inhibited by cellobiose – a potential tool for exploring HTG’s biological functions. Interestingly, cellobiose retarded Equisetum stem elongation, suggesting a role for HTG in growth.
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影响因子：
4.1
作者：
FRY, SC;SMITH, RC;MATTHEWS, KJ
通讯作者：
MATTHEWS, KJ
影响因子：
3.8
作者：
Chormova D;Franková L;Defries A;Cutler SR;Fry SC
通讯作者：
Fry SC
影响因子：
7.4
作者：
HETHERINGTON, PR;FRY, SC
通讯作者：
FRY, SC
影响因子：
7.2
作者：
Frankova, Lenka;Fry, Stephen C.
通讯作者：
Fry, Stephen C.
果皮同质分离剂掩盖了植物细胞壁中丰富的木葡聚糖表位。
影响因子：
5.3
作者：
Marcus SE;Verhertbruggen Y;Hervé C;Ordaz-Ortiz JJ;Farkas V;Pedersen HL;Willats WG;Knox JP
通讯作者：
Knox JP