Biosynthesis of Mannosyl- and Glucosyl-Phosphoryl-Polyprenols in Cotton Fibers

Abstract A particulate enzyme fraction from cotton fibers catalyzes the incorporation of mannose from GDP-[14C]mannose and glucose from UDP-[14C]glucose into acidic lipids. Both of these lipids are retained on DEAE-cellulose and both have mobilities on Silica Gel G in three solvent systems (neutral, basic, and acidic) similar to the mannosyl-1-phosphoryl undecaprenol isolated from micrococci. The sugar moieties of both lipids are very labile to acid hydrolysis and are released in a few minutes in 0.001 to 0.003 n HCl at 100°. Synthesis of the mannolipid is reversed by GDP, while synthesis of the glucolipid is reversed by UDP. Purified [14C]mannolipid incubated with membrane preparations in the presence of GDP gives rise to GDP-[14C]mannose. Similarly [14C]glucolipid in the presence of UDP results in formation of UDP-[14C]glucose. The enzyme preparation was treated with acetone to remove endogenous lipids. This enzyme fraction was inactive in catalyzing mannolipid formation in the absence of added lipid "acceptor." An active lipid fraction was isolated from cotton fibers by extraction with chloroform-methanol and was purified on DEAE-cellulose. The active component has an Rf similar to that reported for ficaprenyl phosphate. Synthetic ficaprenyl phosphate was very effective in replacing the requirement for the active cotton lipid acceptor. The product formed from GDP-[14C]mannose and ficaprenyl phosphate was similar to that formed from GDP-[14C]mannose and endogenous lipids. Various properties of the mannosyltransferase were examined. The enzyme required Mg++ with an optimum concentration being 1.5 x 10-2 m. The Km for GDP-[14C]mannose was estimated to be 6.7 x 10-7 m and the pH optimum in Tris buffer was 7.5. The apparent Km for ficaprenyl phosphate was about 2 to 4 x 10-5 m. Mannolipid, previously characterized as mannosyl-1-phosphoryl undecaprenol (9), was synthesized by extracts of Mycobacterium smegmatis from GDP-[14C]mannose. This purified lipid as well as the purified mannolipid from cotton gave rise to GDP-[14C]mannose when incubated with the particulate enzyme from M. smegmatis. Although GDP-mannose formation was not absolutely dependent on the addition of GDP probably because the particulate enzyme contains this endogenous nucleotide, GDP did stimulate GDP-mannose synthesis. Likewise, both mannolipids were incubated with the particulate enzyme from cotton fibers. In this experiment, GDP-[14C]mannose was formed from both lipids but only in the presence of added GDP. The cotton lipid was three to five times as active in this system. These experiments indicate that the lipid moiety of the cotton glycolipids is a polyprenol but probably differs somewhat from that of mycobacteria. To determine which sugars might be present in acidic lipids of intact cotton bolls, 250 bolls were extracted with chloroform-methanol and the acidic lipids purified on DEAE-cellulose. Sugars were released by mild acid hydrolysis, reduced with NaBH4, converted to their acetate derivatives, and analyzed by gas-liquid chromatography. The major sugars detected were glucose and mannose but significant amounts of arabinose and galactose were also observed as well as three as yet unidentified peaks.