Two isomers of glucosaminylphosphatidylglycerol. Their occurrence in Bacillus megaterium, structural analysis, and chemical synthesis

Abstract

When Bacillus megaterium is harvested in the stationary phase of growth from a medium in which the pH was lowered gradually during growth from 7.2 to 5.0, two isomers of glucosaminylphosphatidylglycerol are present. Both isomers were isolated and characterized using chemical and enzymatic degradation procedures. Selective removal of the glucosamine moieties yielded phosphatidylglycerol which was shown to have the stereochemical configuration 3- sn-phosphatidyl-1′- sn-glycerol. Partial acid hydrolysis of the 2′ and 3′ isomers of glucosaminylphosphatidyl-glycerol gave glucosaminyl-2-glycerol and glucosaminyl-3-glycerol, respectively. Identification of these breakdown products was facilitated by comparison with the two synthetic isomers. Chemical synthesis of the two glucosaminylphosphatidylglycerol isomers was carried out and the properties of the synthetic and the natural phospholipids were compared. In all the chemical and enzymatic degradation studies, the corresponding natural and synthetic compounds appeared to be fully identical and the structures of the two phospholipids were established as 3- sn-phosphatidyl-1′-[3′-(2-amino-2-deoxy-β- d-glucopyranosyl)]- sn-glycerol and 3- sn-phosphatidyl-1′-[2′-(2-amino-2-deoxy-β- d-glucopyranosyl)]- sn-glycerol. Whereas both glucosaminylphosphatidylglycerol isomers are good substrates for phospholipase A, phospholipases C and D are hydrolyzing the 3′ isomer slowly, and the 2′ isomer is hardly degraded by the enzymes. This difference between the two isomers in susceptibility towards phospholipases C and D is correlated with differences in area per molecule as measured in monolayer experiments. Glucosaminyl-2′-phosphatidylglycerol occupied 82 Å 2 per molecule at pH 9 and a pressure of 20 dynes/cm. The 3′ isomer occupied 77 Å 2 per molecule under the same conditions. These data indicate the presence of a large polar headgroup which might interfere with the enzyme-substrate interaction. Glucosaminyl-3′-phosphatidylglycerol accounts for about 12% of the total phospholipids extracted, whereas the amount of the corresponding 2′ isomer is 2 to 3 times higher. The accumulation of the latter compound is favored by a low environmental pH. After cultivation of the cells at neutral pH, this phospholipid could not be detected, whereas the amount of glucosaminyl-3′-phosphatidylglycerol appears to be rather constant.