SYNTHESIS OF ALPHA-1-]6-MANNOOLIGOSACCHARIDES IN MYCOBACTERIUM-SMEGMATIS - FUNCTION OF BETA-MANNOSYLPHOSPHORYLDECAPRENOL AS THE MANNOSYL DONOR

  • 25 December 1989
    • journal article
    • research article
    • Vol. 264  (36) , 21621-21628
Abstract
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.fwdarw.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.fwdarw.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.fwdarw.6-linked mannooligosaccharides as acceptors. The Km values for mannose, methylmannoside, and .alpha.1.fwdarw.6-linked mannobiose were 30-90 mM, whereas for .alpha.1.fwdarw.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.fwdarw.2 linkage to the longer .alpha.1.fwdarw.6-mannooligosaccharides in a reaction that was specific for GDP-mannose as the donor. The membrane preparation also contained an endo-.alpha.1.fwdarw.-6-mannanase activity that degraded products longer than mannotriose by cleavage of trisaccharide units from the nonreducing end of the .alpha.1.fwdarw.6-mannooligosaccharides.