Biosynthesis of mycobacterial methylmannose polysaccharides requires a unique 1- O -methyltransferase specific for 3- O -methylated mannosides

Abstract

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.

S-Adenosylmethionine (SAM)-Dependent Methyltransferase MftM is Responsible for Methylation of the Redox Cofactor Mycofactocin

“…The analysis of cell extracts by liquid chromatography–mass spectrometry (LC–MS) revealed a mass shift for methylmycofactocins of +14.0498 m / z (Figure A,B and Figure S1), suggesting one additional 13 C label derived from l -methionine-(methyl- 13 C) in the glycosyl chain. This result was compatible with the assumption that the O -methylation is catalyzed by S -adenosyl methionine (SAM)-dependent methyltransferase …”

“…This result was compatible with the assumption that the O-methylation is catalyzed by S-adenosyl methionine (SAM)-dependent methyltransferase. 16 The identification of the gene, however, was hampered by the fact that over 100 putative methyltransferases were annotated in the genome of M. smegmatis. From these genes, seven candidates (Table 1) matching one of the following criteria were selected for gene inactivation: (1) transcriptional upregulation in the presence of ethanol, which is known to induce the biosynthesis of mycofactocin, 1,15 (2) proximity to oligosaccharide-modifying gene clusters, or (3) proximity to genes associated with mycofactocin.…”

“…This result was compatible with the assumption that the O -methylation is catalyzed by S -adenosyl methionine (SAM)-dependent methyltransferase. 16 …”

Towards better syntheses of partially methylated carbohydrates?

“…1c) whose biosynthesis requires recently discovered specific enzymes. 8 Therefore, the synthesis of partially methylated oligosaccharides could enable the discovery of vaccine ingredients to prevent various infections. 9–12…”

Microbial Biosynthesis of Chrysazin Derivatives in Recombinant Escherichia coli and Their Biological Activities

“…Methyltransferases are important enzymes in the modification of different substrate. However, methylation of sugar is very rare [ 20 , 21 ]. Generally, transfer of the sugar group is catalyzed by glycosyltransferases (GTs) with activated NDP-sugars as sugar donors.…”