Department of Plant Biology, University of Georgia, Athens, GA 30602, USA.
Department of Chemistry, University of Georgia, Athens, GA 30602, USA.
Plant Cell Physiol. 2017 Dec 1;58(12):2126-2138. doi: 10.1093/pcp/pcx147.
Xylan is a major hemicellulose in the secondary walls of vessels and fibers, and its acetylation is essential for normal secondary wall assembly and properties. The acetylation of xylan can occur at multiple positions of its backbone xylosyl residues, including 2-O-monoacetylation, 3-O-monoacetylation, 2,3-di-O-acetylation and 3-O-acetylation of 2-O-glucuronic acid (GlcA)-substituted xylosyl residues, but the biochemical mechanism controlling the regiospecific acetylation of xylan is largely unknown. Here, we present biochemical characterization of a group of Arabidopsis thaliana DUF231-containing proteins, namely TBL28, ESK1/TBL29, TBL30, TBL3, TBL31, TBL32, TBL33, TBL34 and TBL35, for their roles in catalyzing the regiospecific acetylation of xylan. Acetyltransferase activity assay of recombinant proteins demonstrated that all of these proteins possessed xylan acetyltransferase activities catalyzing the transfer of acetyl groups from acetyl-CoA onto xylooligomer acceptors albeit with differential specificities. Structural analysis of their reaction products revealed that TBL28, ESK1, TBL3, TBL31 and TBL34 catalyzed xylan 2-O- and 3-O-monoacetylation and 2,3-di-O-acetylation with differential positional preference, TBL30 carried out 2-O- and 3-O-monoacetylation, TBL35 catalyzed 2,3-di-O-acetylation, and TBL32 and TBL33 mediated 3-O-acetylation of 2-O-GlcA-substituted xylosyl residues. Furthermore, mutations of the conserved GDS and DXXH motifs in ESK1 were found to result in a complete loss of its acetyltransferase activity. Together, these results establish that these nine DUF231-containing proteins are xylan acetyltransferases mediating the regiospecific acetylation of xylan and that the conserved GDS and DXXH motifs are critical for their acetyltransferase activity.
木聚糖是导管和纤维次生壁中的主要半纤维素，其乙酰化对于正常的次生壁组装和性质至关重要。木聚糖的乙酰化可以发生在其糖基残基的多个位置，包括 2-O-单乙酰化、3-O-单乙酰化、2,3-二-O-乙酰化和 2-O-葡萄糖醛酸（GlcA）取代的木糖基残基的 3-O-乙酰化，但控制木聚糖区域特异性乙酰化的生化机制在很大程度上尚不清楚。在这里，我们对一组拟南芥 DUF231 包含蛋白，即 TBL28、ESK1/TBL29、TBL30、TBL3、TBL31、TBL32、TBL33、TBL34 和 TBL35，进行了生化特性分析，以研究它们在催化木聚糖区域特异性乙酰化中的作用。重组蛋白的乙酰转移酶活性测定表明，所有这些蛋白都具有木聚糖乙酰转移酶活性，能够将乙酰基从乙酰辅酶 A 转移到木寡糖受体上，但具有不同的特异性。对其反应产物的结构分析表明，TBL28、ESK1、TBL3、TBL31 和 TBL34 催化木聚糖 2-O-和 3-O-单乙酰化和 2,3-二-O-乙酰化，具有不同的位置偏好，TBL30 进行 2-O-和 3-O-单乙酰化，TBL35 催化 2,3-二-O-乙酰化，TBL32 和 TBL33 介导 2-O-GlcA 取代的木糖基残基的 3-O-乙酰化。此外，发现 ESK1 中的保守 GDS 和 DXXH 模体的突变导致其乙酰转移酶活性完全丧失。总之，这些结果表明，这九个包含 DUF231 的蛋白是木聚糖乙酰转移酶，介导木聚糖的区域特异性乙酰化，保守的 GDS 和 DXXH 模体是其乙酰转移酶活性的关键。
