Department of Protein Chemistry and Technology, CSIR-Central Food Technological Research Institute, Mysuru, 570 020, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, Uttar Pradesh, India.
Department of Protein Chemistry and Technology, CSIR-Central Food Technological Research Institute, Mysuru, 570 020, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, Uttar Pradesh, India.
Biochem Biophys Res Commun. 2019 Mar 12;510(3):358-363. doi: 10.1016/j.bbrc.2019.01.085. Epub 2019 Feb 1.
Comprehensive knowledge on the role of substrate subsites is a prerequisite to understand the interaction between glycoside hydrolase and its substrate. The present study delineates the role of individual substrate subsites present in ManB-1601 (GH26 endo-mannanase from Bacillus sp.) towards interaction with mannans. Isothermal titration calorimetry of catalytic mutant (E167A/E266A) of ManB-1601 with mannobiose to mannohexose revealed presence of six substrate subsites in ManB-1601. The amino acids present in substrate subsites of ManB-1601 were found to be highly conserved among GH26 endo-mannanases from Bacillus spp. Qualitative substrate binding analysis of subsite mutants by native affinity gel electrophoresis suggested that -3, -2, -1, +1 and + 2 subsites have a major role while, -4 subsite had minor role towards mannan binding. Affinity gels also pointed out the pivotal role of -1 subsite towards glucomannan binding. Quantitative substrate binding analysis using fluorescence titration revealed that -1 and -2 subsite mutants had 27- and 30-fold higher binding affinity (K) for carob galactomannan when compared with catalytic mutant. The -1 subsite mutant also had highest K values for glucomannan (13.6-fold) and ivory nut mannan (5-fold) among all mutants. The positive subsites contributed more towards binding with glucomannan (up to 10-fold higher K) and ivory nut mannan (up to 4.3-fold higher K) rather than carob galactomannan (up to 4-fold higher K). Between distal subsites, -3 mutant displayed 10-fold higher K for both carob galactomannan and glucomannan while, -4 mutant did not show any noticeable change in K values when compared to catalytic mutant.
综合了解酶与底物相互作用中底物亚位的作用是理解糖苷水解酶与其底物相互作用的前提。本研究阐述了 ManB-1601（来源于芽孢杆菌的 GH26 内切甘露聚糖酶）中单个底物亚位在与甘露聚糖相互作用中的作用。利用差示扫描量热法对 ManB-1601 的催化突变体（E167A/E266A）与甘露二糖至甘露六糖的作用进行研究，发现 ManB-1601 中有 6 个底物亚位。研究发现，芽孢杆菌 GH26 内切甘露聚糖酶中 ManB-1601 底物亚位的氨基酸高度保守。通过天然亲和凝胶电泳对亚位突变体的定性底物结合分析表明，-3、-2、-1、+1 和+2 亚位在甘露聚糖结合中起主要作用，而-4 亚位的作用较小。亲和凝胶还指出-1 亚位在魔芋甘露聚糖结合中起关键作用。利用荧光滴定进行定量底物结合分析表明，与催化突变体相比，-1 和-2 亚位突变体对卡拉胶半乳甘露聚糖的结合亲和力（K）分别提高了 27 倍和 30 倍。-1 亚位突变体对魔芋甘露聚糖（提高 13.6 倍）和象牙果甘露聚糖（提高 5 倍）的 K 值也最高。正亚位与魔芋甘露聚糖（K 值提高高达 10 倍）和象牙果甘露聚糖（K 值提高高达 4.3 倍）的结合作用强于卡拉胶半乳甘露聚糖（K 值提高高达 4 倍）。在远位亚位中，-3 突变体对卡拉胶半乳甘露聚糖和魔芋甘露聚糖的 K 值均提高了 10 倍，而-4 突变体与催化突变体相比，K 值没有明显变化。
