Biochemical characterization of a glycosyltransferase Gtf3 from Mycobacterium smegmatis: a case study of improved protein solubilization

Abstract

Glycosyltransferases (GTs) are widely present in several organisms. These enzymes specifically transfer sugar moieties to a range of substrates. The processes of bacterial glycosylation of the cell wall and their relations with host-pathogen interactions have been studied extensively, yet the majority of mycobacterial GTs involved in the cell wall synthesis remain poorly characterized. Glycopeptidolipids (GPLs) are major class of glycolipids present on the cell wall of various mycobacterial species. They play an important role in drug resistance and host-pathogen interaction virulence. Gtf3 enzyme performs a key step in the biosynthesis of triglycosylated GPLs. Here, we describe a general procedure to achieve expression, purification, and crystallization of recombinant protein Gtf3 from Mycobacterium smegmatis using an E. coli expression system. We reported also a combined bioinformatics and biochemical methods to predict aggregation propensity and improve protein solubilization of recombinant Gtf3. NVoy, a carbohydrate-based polymer reagent, was added to prevent protein aggregation by binding to hydrophobic protein surfaces of Gtf3. Using intrinsic tryptophan fluorescence quenching experiments, we also demonstrated that Gtf3-NVoy enzyme interacted with TDP and UDP nucleotide ligands. This case report proposes useful tools for the study of other glycosyltransferases which are rather difficult to characterize and crystallize.

Insertion Mutation of MSMEG_0392 Play an Important Role in Resistance of M. smegmatis to Mycobacteriophage SWU1

“…It plays an important role in bacterial drug resistance and host-pathogen interaction, and also in phage resistance of Mycobacteria. 28 Glycosyltransferases are widely found in mycobacterial cells. These enzymes transfer glycosyl onto a range of substrates and participate in the synthesis of GPLs.…”

Bioinformatic Characterization of a Kappa-Carrageenase from Pseudomonas fluorescens

“…[7]. Moreover, experimental methods aiming third structure determination of proteins are expensive and require a lot of time [8]. However, using in silico enzyme structure analysis tools is a cost-effective and efficient method that can lead to selection of these characterized enzymes and proper use in large-scale industrial production [9].…”