Active site of Zn(2+)-dependent sn-glycerol-1-phosphate dehydrogenase from Aeropyrum pernix K1.
The enzyme sn-glycerol-1-phosphate dehydrogenase (Gro1PDH, EC 1.1.1.261) is key to the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate) of archaeal ether lipids. This enzyme catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-1-phosphate. To date, no information about the active site and catalytic mechanism of this enzyme has been reported. Using the sequence and structural information for glycerol dehydrogenase, we constructed six mutants (D144N, D144A, D191N, H271A, H287A and D191N/H271A) of Gro1PDH from Aeropyrum pernix K1 and examined their characteristics to clarify the active site of this enzyme. The enzyme was found to be a zinc-dependent metalloenzyme, containing one zinc ion for every monomer protein that was essential for activity. Site-directed mutagenesis of D144 increased the activity of the enzyme. Mutants D144N and D144A exhibited low affinity for the substrates and higher activity than the wild type, but their affinity for the zinc ion was the same as that of the wild type. Mutants D191N, H271A and H287A had a low affinity for the zinc ion and a low activity compared with the wild type. The double mutation, D191N/H271A, had no enzyme activity and bound no zinc. From these results, it was clarified that residues D191, H271 and H287 participate in the catalytic activity of the enzyme by binding the zinc ion, and that D144 has an effect on substrate binding. The structure of the active site of Gro1PDH from A. pernix K1 seems to be similar to that of glycerol dehydrogenase, despite the differences in substrate specificity and biological role.
Han
Jin-Suk
JS
Division of Food and Biotechnology, Dongeui Institute of Technology, Busan, Korea.
Ishikawa
Kazuhiko
K
D041522:Aeropyrum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D019641:DNA, Archaeal / Q000235:genetics; D005993:Glycerolphosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D016415:Sequence Alignment; D015032:Zinc / Q000378:metabolism*
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D005993:Glycerolphosphate Dehydrogenase; C098215:glycerol-1-phosphate dehydrogenase; D015032:Zinc