Regulation of phospholipid biosynthesis in Escherichia coli. Cloning of the structural gene for the biosynthetic sn-glycerol-3-phosphate dehydrogenase.
The structural gene for the Escherichia coli biosynthetic sn-glycerol-3-phosphate (glycerol-P) dehydrogenase gpsA, was transferred from a defective transducing phage (lambda dcysE, gpsA) into the Eco RI site of plasmid pMB9 by recombinant DNA techniques. The recombinant plasmids suppressed the glycerol-P requirement of gpsA- mutants and strains bearing one such plasmid, pDC2, overproduced the glycerol-P dehydrogenase about 60-fold. The glycerol-P dehydrogenase from a strain bearing the pDC2 was purified 200-fold to homogeneity. This is contrasted to the 12,000-fold purification required to purify the enzyme from a wild type strain (Edgar, J. R., and Bell, R. M. (1978) J. Biol. Chem. 253, 6348-6353). The homogeneous enzyme purified from a strain bearing the pDC2 plasmide was strongly inhibited by glycerol-P (Ki of 2.5 microM). The introduction of the pDC2 plasmid into glycerol-P auxotrophs containing a Km-defective glycerol-P acyltranferase, defined by the plsB locus, caused a 60-fold overproduction of the glycerol-P requirement. This strongly suggests that the intracellular level of glycerol-P is stringently regulated in vivo by a mechanism involving feedback inhibition of the glycerol-P dehydrogenase by glycerol-P.
D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005796:Genes; D005838:Genotype; D005993:Glycerolphosphate Dehydrogenase / Q000096:biosynthesis* / Q000302:isolation & purification; D009154:Mutation; D010743:Phospholipids / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D013045:Species Specificity; D014158:Transcription, Genetic
D004274:DNA, Recombinant; D010743:Phospholipids; D005993:Glycerolphosphate Dehydrogenase