Komvongsa Juthamath, Luang Sukanya, Marques Joaquim V, Phasai Kannika, Davin Laurence B, Lewis Norman G, Ketudat Cairns James R
School of Biochemistry, Institute of Science, and Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA.
Biochim Biophys Acta. 2015 Jul;1850(7):1405-14. doi: 10.1016/j.bbagen.2015.03.013. Epub 2015 Apr 9.
Rice Os9BGlu31 is a transglucosidase that can transfer glucose to phenolic acids, flavonoids, and phytohormones. Os9BGlu31 displays a broad specificity with phenolic 1-O-β-D-glucose esters acting as better glucose donors than glucosides, whereas the free phenolic acids of these esters are also excellent acceptor substrates.
Based on homology modeling of this enzyme, we made single point mutations of residues surrounding the acceptor binding region of the Os9BGlu31 active site. Products of the wild type and mutant enzymes in transglycosylation of phenolic acceptors from 4-nitrophenyl β-D-glucopyranoside donor were identified and measured by UPLC and negative ion electrospray ionization tandem mass spectrometry (LCMSMS).
The most active variant produced was W243N, while I172T and L183Q mutations decreased the activity, and other mutations at W243 (A, D, M, N, F and Y) had variable effects, depending on the acceptor substrate. The Os9BGlu31 W243N mutant activity was higher than that of wild type on phenolic acids and kaempferol, a flavonol containing 4 hydroxyl groups, and the wild type Os9BGlu31 produced only a single product from each of these acceptors in significant amounts, while W243 variants produced multiple glucoconjugates. Fragmentation analysis provisionally identified the kaempferol transglycosylation products as kaempferol 3-O, 7-O, and 4'-O glucosides and 3,7-O, 4',7-O, and 3,4'-O bis-O-glucosides. The Os9BGlu31 W243 mutants were also better able to use kaempferol 3-O-glucoside as a donor substrate.
The W243 residue was found to be critical to the substrate and product specificity of Os9BGlu31 transglucosidase and mutation of this residue allows production of a range of glucoconjugates.
水稻Os9BGlu31是一种转葡糖苷酶，可将葡萄糖转移至酚酸、类黄酮和植物激素。Os9BGlu31表现出广泛的特异性，酚类1-O-β-D-葡萄糖酯作为葡萄糖供体比糖苷更好，而这些酯的游离酚酸也是优良的受体底物。
基于该酶的同源建模，我们对Os9BGlu31活性位点受体结合区域周围的残基进行了单点突变。通过超高效液相色谱（UPLC）和负离子电喷雾电离串联质谱（LCMSMS）鉴定并测量了野生型和突变型酶在以4-硝基苯基β-D-吡喃葡萄糖苷供体进行酚类受体转糖基化反应中的产物。
产生的活性最高的变体是W243N，而I172T和L183Q突变降低了活性，W243处的其他突变（A, D, M, N, F和Y）根据受体底物的不同产生了不同的影响。Os9BGlu31 W243N突变体在酚酸和山奈酚（一种含4个羟基的黄酮醇）上的活性高于野生型，野生型Os9BGlu31从这些受体中各自仅产生一种大量的单一产物，而W243变体产生了多种葡萄糖共轭物。碎片分析初步鉴定出山奈酚转糖基化产物为山奈酚3-O、7-O和4'-O葡萄糖苷以及3,7-O、4',7-O和3,4'-O双-O-葡萄糖苷。Os9BGlu31 W243突变体也更能利用山奈酚3-O-葡萄糖苷作为供体底物。
发现W243残基对Os9BGlu31转葡糖苷酶的底物和产物特异性至关重要，该残基的突变可产生一系列葡萄糖共轭物。
