Structure Analysis and Site-Directed Mutagenesis of the Glycosyltransferase UGT71B8 Leads to Increased Stability and Substrate Activity in Arabidopsis thaliana.

Abstract

The uridine diphosphate-glycosyltransferase (UGT) family catalyses the glucuronidation of the glycosyl group of a nucleotide sugar to an acceptor compound (substrate), it serves as controlling reaction for bioactivity, storage and decrease toxicity of different compounds in living organisms. UGT71B8 belongs to 71B family of UGTs and its donor sugars are UDP glucose, UDP galactose and UDP 5S glucose, respectively. The current study was designed to induce site-directed mutagenesis (SDM) to investigate the activity in UGT71B8 enzyme. During first step, in silico conformational change through 3D structure model was drawn and it was found that all the amino acids of mutation site were found in allowed region. The relative surface accessibility (RSA) and absolute surface accessibility (ASA) of UGT71B8 were found as 0.042-0.037 and 7.424, respectively, which shows that UGT71B8 T138M remains stable after SDM. This prediction model thus led to the efficacious mutation of UGT71B8 enzyme. Mass spectrometric analysis of UGT71B8T138M showed reduced activity with its substrate UDP glucose and kaempherol as acceptor molecule. Moreover, no new substrate activity of UGT71B8 was found. This data would direct future endeavors to engineer more glycosyltransferases of plants to augment its activity with different substrates and provide a basis for more exploration of UGT71B8 as an active compound for potential anti-cancer therapeutics.