Characterization and localization of phosphatidylglycerophosphate and phosphatidylserine synthases in Rhodobacter sphaeroides.
Catalytic properties and membrane associations of the phosphatidylglycerophosphate (PGP) and phosphatidylserine (PS) synthases of Rhodobacter sphaeroides were examined to further characterize sites of phospholipid biosynthesis. In preparations of cytoplasmic membrane (CM) enriched in these activities, apparent Km values of PGP synthase were 90 microM for sn-glycerol-3-phosphate and 60 microM for CDP-diacylglycerol; the apparent Km of PS synthase for L-serine was near 165 microM. Both enzymes required Triton X-100 with optimal PS synthase activity at a detergent/CDP-diacylglycerol (mol/mol) ratio of 7.5:1.0, while for optimal PGP synthase, a range of 10-50:1.0 was observed. Unlike the enzyme in Escherichia coli and several other Gram-negative bacteria, the PS synthase activity had a specific requirement for magnesium and was tightly associated with membranes rather than ribosomes in crude cell extracts. Sedimentation studies suggested that the PGP synthase was distributed uniformly over the CM in both chemoheterotrophically and photoheterotrophically grown cells, while the PS synthase was confined mainly to a vesicular CM fraction. Solubilized PGP synthase activity migrated as a single band with a pI value near 5.5 in a chromato-focusing column and 5.8 on isoelectric focusing; in the latter procedure, the pI was shifted to 5.3 in the presence of CDP-diacylglycerol. The PGP synthase activity gave rise to a single polypeptide band in lithium dodecyl sulfate-polyacrylamide gel electrophoresis at 4 degrees C.
Radcliffe
C W
CW
Department of Biochemistry, Rutgers University, Piscataway, NJ 08855-1059.
Steiner
F X
FX
Carman
G M
GM
Niederman
R A
RA
D010717:CDPdiacylglycerol-Serine O-Phosphatidyltransferase / Q000032:analysis*; D002499:Centrifugation, Density Gradient; D002500:Centrifugation, Isopycnic; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D010743:Phospholipids / Q000096:biosynthesis; D010770:Phosphotransferases / Q000032:analysis*; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D017855:Transferases (Other Substituted Phosphate Groups)
D010743:Phospholipids; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C020676:CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase; D010717:CDPdiacylglycerol-Serine O-Phosphatidyltransferase