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

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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.

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Acknowledgments

We would like to thank Dr. Yves Bourne from AFMB laboratory facilities, Dr. Badreddine Douzi and Dr. Renaud Vincentelli for support in protein expression and purification, Dr. Silvia Spinelli for crystallization facilities as well as Stéphanie Blangy, and Dr. David Veesler for support in MALS/UV/refractometry/SEC.

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Authors and Affiliations

  1. Department of Science of Nature and Life, Institute of Science, University Center Belhadj Bouchaib of Ain Temouchent, Po Box 284, 46000, Ain Temouchent, Algeria

    Mahfoud Bakli

  2. Laboratory of Physiology, Pathophysiology and Biochemistry of Nutrition, University Abou-Bekr Belkaid of Tlemcen, Tlemcen, Algeria

    Mahfoud Bakli, Nassima Mokhtari-Soulimane & Hafida Merzouk

  3. University of Strasbourg, CNRS, Architecture and Reactivity of RNA, UPR9002, Strasbourg, France

    Loukmane Karim

  4. Architecture et Fonction des Macromolécules Biologiques, CNRS, Aix-Marseille University, Marseille, France

    Mahfoud Bakli & Florence Vincent

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  1. Mahfoud Bakli
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Contributions

MB and FV conceived and designed the experiments. MB performed the experiments. MB, LK, and FV analyzed the data. MB, LK, NMS, HM, and FV wrote the manuscript.

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Correspondence to Mahfoud Bakli.

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Bakli, M., Karim, L., Mokhtari-Soulimane, N. et al. Biochemical characterization of a glycosyltransferase Gtf3 from Mycobacterium smegmatis: a case study of improved protein solubilization. 3 Biotech 10, 436 (2020). https://doi.org/10.1007/s13205-020-02431-x

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