Synthesis of alpha 1----6-mannooligosaccharides in Mycobacterium smegmatis. Function of beta-mannosylphosphoryldecaprenol as the mannosyl donor

Incubation of a membrane fraction from Mycobacterium smegmatis cells with GDP-mannose and free mannose at pH 7 in presence of Mg2+ ions resulted in the formation of a series of alpha 1---6-linked mannooligosaccharides with up to 12 mannoses. The membrane fraction also catalyzed incorporation of mannose from GDP-mannose into a lipid-soluble product with the properties of a mannosyl phospholipid. A similar product was formed by the incubation of the membrane protein with decaprenol phosphate and GDP-mannose, and it was characterized as beta-mannosylphosphoryldecaprenol. A pulse-chase experiment suggested that the mannosyl phospholipid was an intermediate in alpha 1---6-linked mannooligosaccharide synthesis, and the isolated beta-mannosylphosphoryldecaprenol was shown to function as a direct mannosyl donor on incubation with mannose, methyl alpha-D-mannoside, or alpha 1---6-linked mannooligosaccharides as acceptors. The Km values for mannose, methylmannoside, and alpha 1---6-linked mannobiose were 30-90 mM, whereas for alpha 1---6-linked mannotriose, mannotetraose, and mannopentaose the Km dropped to 2 mM. A weak enzymic activity was detected at pH 6 in the presence of both Mg2+ and Mn2+ ions that catalyzed addition of mannose in alpha 1---2 linkage to the longer alpha 1---6-mannooligosaccharides in a reaction that was specific for GDP-mannose as the donor. The membrane preparation also contained an endo-alpha 1---6-mannanase activity that degraded products longer than mannotriose by cleavage of trisaccharide units from the nonreducing end of the alpha 1---6-mannooligosaccharides.