Plant Tissue Culture Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, Maharashtra, 411008, India.
Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, People's Republic of China.
World J Microbiol Biotechnol. 2018 Sep 25;34(10):150. doi: 10.1007/s11274-018-2534-4.
This work used an approach of enzyme engineering towards the improved production of baicalin as well as alteration of acceptor and donor substrate preferences in UGT73A16. The 3D model of Withania somnifera family-1 glycosyltransferase (UGT73A16) was constructed based on the known crystal structures of plant UGTs. Structural and functional properties of UGT73A16 were investigated using docking and mutagenesis. The docking studies were performed to understand the key residues involved in substrate recognition. In the molecular model of UGT73A16, substrates binding pockets are located between N- and C-terminal domains. Modeled UGT73A16 was docked with UDP-glucose, UDP-glucuronic acid (UDPGA), kaempferol, isorhamnetin, 3-hydroxy flavones, naringenin, genistein and baicalein. The protein-ligand interactions showed that His 16, Asp 246, Lys 255, Ala 337, Gln 339, Val 340, Asn 358 and Glu 362 amino acid residues may be important for catalytic activity. The kinetic parameters indicated that mutants A337C and Q339A exhibited 2-3 fold and 6-7 fold more catalytic efficiency, respectively than wild type, and shifted the sugar donor specificity from UDP-glucose to UDPGA. The mutant Q379H displayed large loss of activity with UDP-glucose and UDPGA strongly suggested that last amino acid residue of PSPG box is important for glucuronosylation and glucosylation and highly specific to sugar binding sites. The information obtained from docking and mutational studies could be beneficial in future to engineer this biocatalyst for development of better ones.
这项工作采用了酶工程的方法，旨在提高黄芩苷的产量，并改变 UGT73A16 的受体和供体底物偏好。根据已知的植物 UGT 晶体结构，构建了茄科毛曼陀罗家族 1 糖基转移酶（UGT73A16）的 3D 模型。利用对接和突变研究了 UGT73A16 的结构和功能特性。对接研究旨在了解参与底物识别的关键残基。在 UGT73A16 的分子模型中，底物结合口袋位于 N-和 C-末端结构域之间。将建模的 UGT73A16 与 UDP-葡萄糖、UDP-葡萄糖醛酸（UDPGA）、山柰酚、异鼠李素、3-羟基黄酮、柚皮苷、染料木黄酮和黄芩苷对接。蛋白-配体相互作用表明，His16、Asp246、Lys255、Ala337、Gln339、Val340、Asn358 和 Glu362 氨基酸残基可能对催化活性很重要。动力学参数表明，突变体 A337C 和 Q339A 的催化效率分别比野生型提高了 2-3 倍和 6-7 倍，并且将糖供体特异性从 UDP-葡萄糖转移到 UDPGA。突变体 Q379H 对 UDP-葡萄糖和 UDPGA 的活性损失很大，这强烈表明 PSPG 框的最后一个氨基酸残基对葡萄糖醛酸化和葡萄糖化很重要，并且对糖结合位点具有高度特异性。对接和突变研究获得的信息可能有助于未来对这种生物催化剂进行工程改造，以开发更好的生物催化剂。
