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Biosynthesis of Mycobacterial Methylmannose Polysaccharides Requires a Unique 1-O-Methyltransferase Specific for 3-O-Methylated Mannosides

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Biosynthesis of Mycobacterial Methylmannose Polysaccharides Requires a Unique 1-O-Methyltransferase Specific for 3-O-Methylated Mannosides Biosynthesis of mycobacterial methylmannose polysaccharides requires a unique 1-O-methyltransferase specific for 3-O-methylated mannosides Jorge Ripoll-Rozadaa,b,1, Mafalda Costac,d,1, José A. Mansoa,b, Ana Maranhac, Vanessa Mirandae, André Sequeirae, M. Rita Venturae, Sandra Macedo-Ribeiroa,b, Pedro José Barbosa Pereiraa,b,2, and Nuno Empadinhasc,f,2 aInstituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, 4200-135 Porto, Portugal; bInstituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal; cCenter for Neuroscience and Cell Biology (CNC), University of Coimbra, 3004-504 Coimbra, Portugal; dPhD Program in Experimental Biology and Biomedicine (PDBEB), Institute for Interdisciplinary Research (IIIUC), University of Coimbra, 3030-789 Coimbra, Portugal; eBioorganic Chemistry Group, Instituto de Tecnologia Química Biológica, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal; and fInstitute for Interdisciplinary Research (IIIC), University of Coimbra, 3030-789 Coimbra, Portugal Edited by Chi-Huey Wong, Academia Sinica, Taipei, Taiwan, and approved December 4, 2018 (received for review August 3, 2018) Mycobacteria are a wide group of organisms that includes strict bacteria also produce a number of intracellular glycoconjugates, pathogens, such as Mycobacterium tuberculosis, as well as environ- including rare polymethylated polysaccharides (PMPSs), which can mental species known as nontuberculous mycobacteria (NTM), some be divided in two classes: 6-O-methylglucose lipopolysaccharides of which—namely Mycobacterium avium—are important opportu- (MGLP) and 3-O-methylmannose polysaccharides (MMP). While nistic pathogens. In addition to a distinctive cell envelope mediating MGLP have been found in all mycobacterial species examined thus critical interactions with the host immune system and largely respon- far and in other related bacteria of the Nocardia genus, MMP are sible for their formidable resistance to antimicrobials, mycobacteria absent from species of the Mycobacterium tuberculosis complex, synthesize rare intracellular polymethylated polysaccharides impli- having a scattered distribution across mycobacteria and an appar- cated in the modulation of fatty acid metabolism, thus critical players ently biased association to rapidly growing species (5). in cell envelope assembly. These are the 6-O-methylglucose Although their physiological functions are still not fully understood, lipopolysaccharides (MGLP) ubiquitously detected across the PMPSs have been proposed to form stable, thermodynamically BIOCHEMISTRY Mycobacterium genus, and the 3-O-methylmannose polysaccha- favored complexes with long-chain fatty acids and palmityol-CoA, rides (MMP) identified only in NTM. The polymethylated nature of these polysaccharides renders the intervening methyltrans- Significance ferases essential for their optimal function. Although the knowl- edge of MGLP biogenesis is greater than that of MMP biosynthesis, Mycobacteria are priority pathogens in terms of drug resistance the methyltransferases of both pathways remain uncharacterized. worldwide and efforts aimed at deciphering their unique meta- Here, we report the identification and characterization of a unique bolic pathways and unveiling new targets for innovative drugs S-adenosyl-L-methionine–dependent sugar 1-O-methyltransferase should be intensified. Mycobacterial polymethylated polysac- (MeT1) from Mycobacterium hassiacum that specifically blocks charides, 6-O-methylglucose lipopolysaccharides (MGLP) and 3-O- the 1-OH position of 3,3′-di-O-methyl-4α-mannobiose, a probable methylmannose polysaccharides (MMP), identified half a century early precursor of MMP, which we chemically synthesized. The high- ago, have been implicated in the metabolism of precursors of cell resolution 3D structure of MeT1 in complex with its exhausted co- envelope lipids crucial for stress resistance and pathogenesis. factor, S-adenosyl-L-homocysteine, together with mutagenesis Although the functions of MGLP and MMP remain to be con- studies and molecular docking simulations, unveiled the enzyme’s firmed experimentally, their tight interactions with fatty acids are reaction mechanism. The functional and structural properties of intrinsically associated to unique and extensive methylation this unique sugar methyltransferase further our knowledge of patterns, resulting from the action of hitherto uncharacterized MMP biosynthesis and provide important tools to dissect the role methyltransferases. Herein, we identify and characterize func- of MMP in NTM physiology and resilience. tionally and structurally a rare sugar methyltransferase for spe- cific methylation of the MMP reducing end, shedding light onto Mycobacterium | polymethylated polysaccharides | sugar an unexplored mycobacterial pathway. methyltransferase | S-adenosyl-L-methionine | 3D structure Author contributions: J.R.-R., M.R.V., S.M.-R., P.J.B.P., and N.E. designed research; J.R.-R., lthough water sanitation is among the greatest health 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., Aachievements of all time, opportunistic nontuberculous 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. mycobacteria (NTM) persist as ubiquitous residents of drinking water globally, including high-income countries. NTM are pre- The authors declare no conflict of interest. dominantly environmental saprophytes whose association with This article is a PNAS Direct Submission. opportunistic infection has steadily increased in recent decades Published under the PNAS license. (1). Some risk factors predisposing to NTM disease are associated 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 with host immune fragilities, although reports of infections in ap- data have been deposited in the Small Angle Scattering Biological Data Bank https:// parently immunocompetent individuals are also on the rise (2). As sasbdb.org (SASBDB entry SASDDJ6); and the X-ray diffraction images have been depos- the growing incidence of NTM disease appears to correlate with a ited 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/ high prevalence of chronic diseases, understanding NTM biology, SBGRID/599, and 10.15785/SBGRID/595). namely how they build and uphold their unique cell envelope, a 1J.R.-R. and M.C. contributed equally to this work. remarkable barrier to antibiotics, is a global health priority. 2To whom correspondence may be addressed. Email: [email protected] or numenius@ Mycobacteria are rich in extracellular cell wall-anchored carbo- cnc.uc.pt. hydrates that, together with diverse complex lipids and lipoglycans, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. confer on them pathogenic properties and an effective shield from 1073/pnas.1813450116/-/DCSupplemental. host immunity (3, 4). In addition to these carbohydrates, myco- Published online January 3, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1813450116 PNAS | January 15, 2019 | vol. 116 | no. 3 | 835–844 Downloaded by guest on September 26, 2021 potentially protecting their hydrophobic chains from degradation by cytoplasmic thioesterases (6–8). PMPSs have also been described as modulators of fatty acid biosynthesis by facilitating the release of the long-chain acyl-CoA products from fatty acid synthase I, thus preventing product inhibition (4). It was initially proposed that fatty acyl-CoAs can induce a helical conformation in PMPSs, favoring the formation of a methyl group-lined hy- drophobic channel where the apolar acyl-CoA ligand is accom- modated (8, 9). Although this postulated architecture of MMP– lipid tight interactions has been recently challenged, a possible biological role in storage of fatty acid precursors for the diverse mycobacterial lipids remains undisputed (10). These properties of PMPSs likely impact the assembly of cell envelope lipids, namely mycolic acids, distinctive components of the mycomembrane (11). Identification of key biosynthetic genes allowed the construction of Mycobacterium smegmatis mutants deficient in MGLP and in which the levels of fatty acids remained unaltered, possibly due to the presence of the apparently isofunctional MMP (12, 13). Still, a role for MGLP in the maintenance of the mycomembrane under temperature stress or other conditions requiring abrupt lipid synthesis or turnover was anticipated. MMP exists as a mixture of linear chains of 11–14 units of α-1,4– linked 3-O-methyl-D-mannose, which unlike the abundant β-1,4– linked mannans have not been identified thus far in biological systems, emphasizing the chemical and structural exceptionality of MMP and the uniqueness of its biosynthetic enzymes (14). The terminal residue at the nonreducing end is unsubstituted, while the reducing end is blocked with an α-methyl aglycon (Fig. 1A)(14).A similar polysaccharide of 11 units of α-1,4–linked 3-O-methyl-D- mannose has been identified in Streptomyces griseus (15, 16) but in this unique structure some units were acetylated at O-6 positions, a feature common to MGLP. Recently, the wastewater bacterium Oligotropha carboxidovorans was also found to synthesize a related polysaccharide of 35–40 3-O-methyl-D-mannose units, but in this case assembled with α-1,2 linkages
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