Abstract
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.
Highlights
The structural gene f o r the Escherichia coli biosyn- (5).The physiological significance of the glycerol-P feedback thetic sn-glycerol-3-phosphate dehydro- inhibition in the regulation of glycerol-P levels was suggested genase, @SA, was transferred from a defective trans- bysuppression of the plsB defect by a feedback-resistant ducing phage (XdcysE, gpsA) into the Eco R I site of mutant in the glycerol-P dehydrogenase, presumably by eleplasmid pMB9 by recombinant DNA techniques
Recombinant plasmids suppressed the glycerol-P re- Recently, the wild type and feedback-resistant glycerol-P
The glycerol-P dehydrogenase from a strain bearing the pDC2 plasmid 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
Summary
The biosynthetic sn-glycerol-3-phosphate (glycerol-P) de- Assay a n d Purification of Glycerol-€‘ Dehydrogenase-Glycerolhydrogenase( called glycerol-P synthase)(EC 1.1.1.8) P dehydrogenase activitywas determined as described previously (7), catalyzes the NAD(P)H-dependent reduction of dihydroxy- using 0.1 mM NADPH and 1.0 mM dihydroxyacetone-P, unless othacetone-P toglycerol-P ( l ) ,a precursor required for phospho- erwise noted. Glycerol-P dehydrogenase was purified as previously lipid biosynthesis in Escherichia coli (2, 3). Transductions were performed as described by Shimada et al (14) Regulation of Phospholipid Biosynthesis except that the Mi2' concentrationin the transduction mixture was increased to 10 mM This was found to enhance the transduction efficiency of AdcysE gpsA by 10- to 100-fold.Heat induction of strain. Recombinant DNA Methods-Purified XdcysE gpsA was suspended in SM buffer a t 3 X 10" particles/ml, sodium dodecyl sulfate was added to 1%(w/v), and thesuspension was incubated a t 65'C for
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