EXPRESSION AND USE OF METHANOBACTERIUM-THERMOAUTOTROPHICUM SN-GLYCEROL 1-PHOSPHATE DEHYDROGENASE FOR THE ASSAY OF SN-GLYCEROL 1-PHOSPHATE IN ARCHAEA

sn-Glycerol-l-phosphate (G-1-P) dehydrogenase is the key enzyme for bi osynthesis of the enantiomeric glycerophosphate backbone of ether phos pholipids of Archaea. The gene encoding this enzyme in Methanobacteriu m thermoautotrophicum (egsA) was used to construct an expression plasm id pTrcG1Pdh for Escherichia coli. The G-1-P dehydrogenase activity of E. coli XL1-blue/pTrcG1Pdh was maximal 8-10 h after induction. The ex pressed G-1-P dehydrogenase was purified 4300-fold from the soluble fr action to homogeniety after 4300 times purification by a procedure con sisting of fractionation with ammonium sulfate precipitation, and hydr ophobic and ion-exchange chromatographies. The yield was about 70%. Th e V-max value for the forward reaction to produce G-1-P was 740 units (mu mol/min)/mg, with a K-m of 0.21 mM for NADH and 0.39 mM for dihydr oxyacetone phosphate. The K-m's for G-I-P and NAD(+) in the backward r eaction were 10.5 and 0.46 mM, respectively. These kinetic constants a re similar to those for the enzyme from M. thermoautotrophicum. G-I-P dehydrogenase was successfully used to analyze the stereospecificity o f glycerophosphate, which is an intermediate of phospholipid biosynthe sis and glycerol metabolism; the rate of NADH formation was proportion al to the G-1-P concentration up to 3 mM in the presence of 0.02 unit of the purified enzyme.