Cloning and site directed mutagenesis of UGT76E1 leads to changed substrate activity in Arabidopsis thaliana

Abstract

Glycosyltransferases are ubiquitous enzymes play vital role in numerous aspects of living organisms and are involved in secondary metabolism. Also known as uridine diphosphate (UDP) glycosyltransferases (UGTs) are engaged in detoxification in living organisms. UGT76E1 is a member of 76E family of UGT comprising of 453 amino acids. The gene for glycosyltransferase, UGT76E1 from Arabidopsis thaliana was expressed and purified through affinity chromatography.Site directed mutagenesis was performed to change hydrophilic threonine (T) into hydrophobic alanine (A) at 134 position. Afterwards the activity of mutant enzyme was analyzed through mass spectrometry. Novobiocin and kaempherol were used as acceptor moleculesfor activity tests. After mutagenesis the mutant UGT76E1T134A lost its activity with its donor sugar UDP glucose, as no peak was observed for the required products glc-novobiocin and glc-kaempherol at 773 and 447 in mass spectrum respectively. The mutant did not attainany new activity with UDP rhamnose also. Complete loss of activity of UGT76E1T134A with UDP glucose as donor sugar suggested for the presence of significant peptides at the site of mutation.The current results showed that glycosyltransferases can be modified to use different substrates by site directed mutagenesis. This UGT enzyme modification could open new horizon in the development of new drugs for cancer treatment.