Expression and use of Methanobacterium thermoautotrophicum sn-glycerol 1-phosphate dehydrogenase for the assay of sn-glycerol 1-phosphate in Archaea

Abstract

sn-glycerol-1-phosphate (G-1-P) dehydrogenase is the key enzyme for biosynthesis of the enantiomeric glycerophosphate backbone of ether phospholipids of Archaea. The gene encoding this enzyme in Methanobacterium thermoautotrophicum ( egsA) was used to construct an expression plasmid pTrcG1Pdh for Escherichia coli. The G-1-P dehydrogenase activity of E. coli XL1-blue/pTrcG1Pdh was maximal 8–10 h after induction. The expressed G-1-P dehydrogenase was purified 4300-fold from the soluble fraction to homogeniety after 4300 times purification by a procedure consisting of fractionation with ammonium sulfate precipitation, and hydrophobic and ion-exchange chromatographies. The yield was about 70%. The V max value for the forward reaction to produce G-1-P was 740 units (μmol/min)/mg, with a K m of 0.21 mM for NADH and 0.39 mM for dihydroxyacetone phosphate. The K m's for G-1-P and NAD + in the backward reaction were 10.5 and 0.46 mM, respectively. These kinetic constants are similar to those for the enzyme from M. thermoautotrophicum. G-1-P dehydrogenase was successfully used to analyze the stereospecificity of glycerophosphate, which is an intermediate of phospholipid biosynthesis and glycerol metabolism; the rate of NADH formation was proportional to the G-1-P concentration up to 3 mM in the presence of 0.02 unit of the purified enzyme.