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J. Biol. Chem., Vol. 264, Issue 36, 21621-21628, Dec, 1989

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

K Yokoyama and CE Ballou
Department of Biochemistry, University of California, Berkeley 94720.

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.
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