Purification and properties of sn-glycerol-1-phosphate dehydrogenase from Methanobacterium thermoautotrophicum: characterization of the biosynthetic enzyme for the enantiomeric glycerophosphate backbone of ether polar lipids of Archaea.
The enzyme which seems to be responsible for the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate) of archaeal ether lipids was purified from a methanogenic archaeon, Methanobacterium thermoautotrophicum, and characterized. The enzyme, sn-glycerol-1-phosphate: NAD(P)+ oxidoreductase (sn-glycerol-1-phosphate dehydrogenase), was purified 7,600-fold from a cell free extract by ammonium sulfate fractionation and seven steps of chromatography. The final preparation exhibited a specific activity of 617 micromol/min/mg (Vmax) and gave a single band corresponding to 38 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The native enzyme showed an apparent molecular mass of 302 kDa on gel-filtration chromatography, indicating it is present as a homooctamer. Maximum activity was observed at 75 degrees C at near neutral pH. The activity was stimulated by potassium ions. The Km for dihydroxyacetone phosphate was 7.5 times smaller than that for sn-glycerol-1-phosphate, suggesting that the formation of sn-glycerol-1-phosphate is the natural direction in the cell. Under the assay conditions used, no product inhibition was observed. The N-terminal amino acid sequence was determined.
Nishihara
M
M
Department of Chemistry, University of Occupational and Environmental Health, Japan, Kitakyushu.
Koga
Y
Y
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology*; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D005993:Glycerolphosphate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism*; D005994:Glycerophosphates / Q000096:biosynthesis; D007700:Kinetics; D017014:Methanobacterium / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D010742:Phospholipid Ethers / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005994:Glycerophosphates; D010742:Phospholipid Ethers; D005993:Glycerolphosphate Dehydrogenase