Differential inhibition of GH family 11 endo-xylanase by rice xylanase inhibitor and verification by a modified yeast two-hybrid system.
Differential inhibition of GH family 11 endo-xylanase by rice xylanase inhibitor and verification by a modified yeast two-hybrid system.
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DOI：
10.1016/j.ijbiomac.2019.04.001
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发表时间：
2019-04
影响因子：
8.2
通讯作者：
Ming-qi Liu;Xiaoqian Wu;Wen-kang Huo;Jiayi Li;X. Weng;Jiaming Liu;Zhiyu Fang
中科院分区：
文献类型：
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作者：
Ming-qi Liu;Xiaoqian Wu;Wen-kang Huo;Jiayi Li;X. Weng;Jiaming Liu;Zhiyu Fang
水稻木聚糖酶抑制剂（RIXI）是一种保护水稻细胞免受病原菌侵害的xip型木聚糖酶抑制剂蛋白。RIXI抑制了大多数微生物木聚糖酶，从而减少了它们的实际应用。重组RIXI对几种家族11内切木聚糖酶具有明显的抑制活性。与rexi在50 °C作用40 min后，reBaxA50、reBaxA、TfxA_CD214和TfxA_CD的残留活性分别为55.6%、30.3%、30.09%和11.20%。reBaxA50和TfxA_CD214与repxi相互作用后，其固有荧光被静态猝灭。rexi降低了reBaxA50和TfxA_CD214对山毛榉木聚糖的水解。分子动力学模拟显示RIXI的长环（残基144-153）插入到11族木聚糖酶的催化裂口中。原生PAGE结果显示，在试管中相互作用后形成了rixi -木聚糖酶复合物。在活酵母细胞中也观察到RIXI和木聚糖酶之间的相互作用。抑制活性测定和改性酵母双杂交结果表明，RIXI对11家族木聚糖酶的抑制活性随着RIXI -木聚糖酶复合物相互作用强度的增加而提高，两者呈正相关。该修饰酵母双杂交体系结构简单，成本低，可推广到其他蛋白质相互作用的研究中。
Rice xylanase inhibitor (RIXI) is a XIP-type xylanase inhibitor protein that protects rice cells from pathogenic organisms. RIXI inhibits most microbial xylanases and thus decreases their practical application. The recombinant RIXI (rePRIXI) showed evident inhibitory activities against several family 11endo-xylanases. After interaction with rePRIXI at 50 °C for 40 min, the residual activities of reBaxA50, reBaxA, TfxA_CD214, and TfxA_CD were 55.6%, 30.3%, 30.09%, and 11.20%, respectively. Intrinsic fluorescence of reBaxA50 and TfxA_CD214 was statically quenched after interaction with rePRIXI. rePRIXI decreased hydrolysis of beechwood xylan by reBaxA50 and TfxA_CD214. Molecular dynamics simulations revealed the long loop (residues 144–153) of RIXI inserts into the catalytic cleft of family 11 xylanases. Native PAGE results revealed the formation of RIXI–xylanase complex after their interaction in the test tube. Interactions were also observed between RIXI and xylanases in living yeast cells. The results of inhibitory activity assay and modified yeast two-hybrid revealed that the inhibitory activity of RIXI on family 11 xylanase improved with the interaction strength of the RIXI–xylanase complex, indicating their positive correlation. The modified yeast two-hybrid system is relatively simple and has low cost, and its use may be extended to other studies on protein–protein interactions.