Kulkarni Tejas S, Khan Samiullah, Villagomez Rodrigo, Mahmood Tahir, Lindahl Sofia, Logan Derek T, Linares-Pastén Javier A, Nordberg Karlsson Eva
Biotechnology, Department of Chemistry, Lund University, Lund, SE-221 00, Sweden.
Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, 45320, Pakistan.
Proteins. 2017 May;85(5):872-884. doi: 10.1002/prot.25256. Epub 2017 Mar 8.
The β-glucosidase TnBgl1A catalyses hydrolysis of O-linked terminal β-glycosidic bonds at the nonreducing end of glycosides/oligosaccharides. Enzymes with this specificity have potential in lignocellulose conversion (degrading cellobiose to glucose) and conversion of bioactive flavonoids (modification of glycosylation results in modulation of bioavailability). Previous work has shown TnBgl1A to hydrolyse 3, 4' and 7 glucosylation in flavonoids, and although conversion of 3-glucosylated substrate to aglycone was low, it was improved by mutagenesis of residue N220. To further explore structure-function relationships, the crystal structure of the nucleophile mutant TnBgl1A-E349G was determined at 1.9 Å resolution, and docking studies of flavonoid substrates were made to reveal substrate interacting residues. A series of single amino acid changes were introduced in the aglycone binding region [N220(S/F), N221(S/F), F224(I), F310(L/E), and W322(A)] of the wild type. Activity screening was made on eight glucosylated flavonoids, and kinetic parameters were monitored for the flavonoid quercetin-3-glucoside (Q3), as well as for the model substrate para-nitrophenyl-β-d-glucopyranoside (pNPGlc). Substitution by Ser at N220 or N221 increased the catalytic efficiency on both pNPGlc and Q3. Residue W322 was proven important for substrate accomodation, as mutagenesis to W322A resulted in a large reduction of hydrolytic activity on 3-glucosylated flavonoids. Flavonoid glucoside hydrolysis was unaffected by mutations at positions 224 and 310. The mutations did not significantly affect thermal stability, and the variants kept an apparent unfolding temperature of 101°C. This work pinpoints positions in the aglycone region of TnBgl1A of importance for specificity on flavonoid-3-glucosides, improving the molecular understanding of activity in GH1 enzymes. Proteins 2017; 85:872-884. © 2016 Wiley Periodicals, Inc.
β-葡萄糖苷酶TnBgl1A催化糖苷/寡糖非还原端O-连接的末端β-糖苷键的水解。具有这种特异性的酶在木质纤维素转化（将纤维二糖降解为葡萄糖）和生物活性黄酮类化合物的转化（糖基化修饰导致生物利用度的调节）方面具有潜力。先前的研究表明TnBgl1A可水解黄酮类化合物中的3、4'和7位糖基化，尽管3-糖基化底物向苷元的转化效率较低，但通过对N220残基进行诱变可提高转化效率。为了进一步探索结构-功能关系，以1.9 Å的分辨率测定了亲核突变体TnBgl1A-E349G的晶体结构，并对黄酮类底物进行对接研究以揭示与底物相互作用的残基。在野生型的苷元结合区域[N220(S/F)、N221(S/F)、F224(I)、F310(L/E)和W322(A)]引入了一系列单氨基酸变化。对八种糖基化黄酮类化合物进行了活性筛选，并监测了黄酮类化合物槲皮素-3-葡萄糖苷（Q3）以及模型底物对硝基苯基-β-D-吡喃葡萄糖苷（pNPGlc）的动力学参数。用丝氨酸取代N220或N221可提高对pNPGlc和Q3的催化效率。已证明残基W322对于底物容纳很重要，因为将其突变为W322A会导致对3-糖基化黄酮类化合物的水解活性大幅降低。黄酮类葡萄糖苷水解不受224和310位突变的影响。这些突变对热稳定性没有显著影响，变体的表观解折叠温度保持在101°C。这项工作确定了TnBgl1A苷元区域中对黄酮类-3-葡萄糖苷特异性重要的位置，增进了对GH1酶活性的分子理解。《蛋白质》2017年；85:872 - 884。© 2016威利期刊公司
