Edited by Chi-Huey Wong, Academia Sinica, Taipei, Taiwan, and approved December 4, 2018 (received for review August 3, 2018)
Mycobacteria are priority pathogens in terms of drug resistance worldwide and efforts aimed at deciphering their unique metabolic pathways and unveiling new targets for innovative drugs should be intensified. Mycobacterial polymethylated polysaccharides, 6-O-methylglucose lipopolysaccharides (MGLP) and 3-O-methylmannose polysaccharides (MMP), identified half a century ago, have been implicated in the metabolism of precursors of cell envelope lipids crucial for stress resistance and pathogenesis. Although the functions of MGLP and MMP remain to be confirmed experimentally, their tight interactions with fatty acids are intrinsically associated to unique and extensive methylation patterns, resulting from the action of hitherto uncharacterized methyltransferases. Herein, we identify and characterize functionally and structurally a rare sugar methyltransferase for specific methylation of the MMP reducing end, shedding light onto an unexplored mycobacterial pathway.
Mycobacteria are a wide group of organisms that includes strict pathogens, such as Mycobacterium tuberculosis, as well as environmental species known as nontuberculous mycobacteria (NTM), some of which—namely Mycobacterium avium—are important opportunistic pathogens. In addition to a distinctive cell envelope mediating critical interactions with the host immune system and largely responsible for their formidable resistance to antimicrobials, mycobacteria synthesize rare intracellular polymethylated polysaccharides implicated in the modulation of fatty acid metabolism, thus critical players in cell envelope assembly. These are the 6-O-methylglucose lipopolysaccharides (MGLP) ubiquitously detected across the Mycobacterium genus, and the 3-O-methylmannose polysaccharides (MMP) identified only in NTM. The polymethylated nature of these polysaccharides renders the intervening methyltransferases essential for their optimal function. Although the knowledge of MGLP biogenesis is greater than that of MMP biosynthesis, the methyltransferases of both pathways remain uncharacterized. Here, we report the identification and characterization of a unique S-adenosyl-l-methionine–dependent sugar 1-O-methyltransferase (MeT1) from Mycobacterium hassiacum that specifically blocks the 1-OH position of 3,3′-di-O-methyl-4α-mannobiose, a probable early precursor of MMP, which we chemically synthesized. The high-resolution 3D structure of MeT1 in complex with its exhausted cofactor, S-adenosyl-l-homocysteine, together with mutagenesis studies and molecular docking simulations, unveiled the enzyme’s reaction mechanism. The functional and structural properties of this unique sugar methyltransferase further our knowledge of MMP biosynthesis and provide important tools to dissect the role of MMP in NTM physiology and resilience.
↵1J.R.-R. and M.C. contributed equally to this work.
Author contributions: J.R.-R., M.R.V., S.M.-R., P.J.B.P., and N.E. designed research; J.R.-R., M.C., J.A.M., A.M., V.M., A.S., P.J.B.P., and N.E. performed research; J.R.-R., M.C., J.A.M., A.M., M.R.V., S.M.-R., P.J.B.P., and N.E. analyzed data; and J.R.-R., P.J.B.P., and N.E. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, https://www.wwpdb.org (PDB ID codes 6H40, 6G7D, 6G80); the SAXS data have been deposited in the Small Angle Scattering Biological Data Bank https://sasbdb.org (SASBDB entry SASDDJ6); and the X-ray diffraction images have been deposited in the SBGrid Database, https://data.sbgrid.org (SBGrid Database entries 10.15785/SBGRID/593, 10.15785/SBGRID/594, 10.15785/SBGRID/597, 10.15785/SBGRID/598, 10.15785/SBGRID/599, and 10.15785/SBGRID/595).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1813450116/-/DCSupplemental.
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