Active site of Zn(2+)-dependent sn-glycerol-1-phosphate dehydrogenase from Aeropyrum pernix K1

Abstract

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.

Figure 1.

Amino acid alignment of several glycerol dehydrogenases (GroDHs) together with sn-glycerol-1-phosphate dehydrogenase (Gro1PDH) of Aeropyrum pernix K1. Archaeal Gro1PDH: Methanobacterium thermoautotrophicum (M. thermo; 370 aa); Pyrococcus abyssi (P. abyssi; 346 aa); and Sulfolobus solfataricus (S. solfa; 351 aa). Bacterial or eukaryotic GroDH: Bacillus stearothermophilus (B. stearo; 370 aa); Escherichia coli (E. coli; 380 aa); and Schizosaccharomyces pombe (S. pombe; 450 aa). Amino acid residues that are identical in all sequences are shaded in dark gray, and residues that are identical in all Gro1PDHs are shaded in light gray. Asterisks indicate amino acid residues changed by site-directed mutagenesis. The box indicates the NAD(P)H binding site.

Figure 2.

Thermal unfolding curves for the wild type and mutant sn-glycerol-1-phosphate dehydrogenase (Gro1PDH) (0.21–0.34 mg ml^–1) at 222 nm of CD. Abbreviation: θ = ellipticity.

Figure 3.

Restoration of activity by zinc ions in sn-glycerol-1-phosphate (Gro1PDH). The Gro1PDH was dialyzed against 50 mM EDTA (pH 8.0) for 24 h at 4 °C, and then against 50 mM Tris-HCl buffer/0.15 M NaCl (pH 8.0). Activity was measured after incubation in 50 mM Tris-HCl buffer/0.15 M NaCl (pH 8.0) containing ZnCl₂ at 4 °C for 1 h. (a) Wild type (○), D144N (▽) and D144A (□). (b) Wild type (●), D191N (▼), H271A (▲) and H287A (■).