PMC

Surface charge comparison of NAD⁺-dependent G3PDH from L. mexicana (A) and NADP⁺ dependent G3PDH from A. fulgidus (B). The surface potential maps were calculated in the presence of 100 mM salt. This figure was prepared with GRASP ().

Alignment of the primary structures of G3PDH from A. fulgidus and L. mexicana. The residues, which are involved in substrate binding including the dinucleotide binding site GXGXXG (residues 7,9,12), are depicted in red. The conserved substrate binding sites are highlighted in green. The secondary structure assignment of G3PDH from A. fulgidus is shown above the sequence alignment.

Structure of NADP⁺-dependent G3PDH from A. fulgidus. (A) Ribbon diagram of the G3PDH dimer when viewed perpendicular to the twofold noncrystallographic axis. One monomer is colored in green, one in blue. The N-terminal dinucleotide binding domains are shown in light green and in light blue; the C-terminal helix domains are shown in dark green and dark blue. The figure was produced with MOLSCRIPT () and Raster3D (). (B) Fold topology diagram of the G3PDH monomer.

Effect of salts on the thermostability of NADP⁺-dependent G3PDH from A. fulgidus. (○) 5 mM K₂HPO₄, (▵) 50 mM K₂HPO₄, (□) 200 mM K₂HPO₄, (•) 50 mM (NH₄)₂SO₄. The pH was 7.0. Aliquots of 0.1 mg enzyme/mL were incubated in sealed tubes for 30 min at the indicated temperatures. After incubation, all tubes were rapidly cooled in an ice bath and analyzed for activity by following NADPH oxidation with dihydroxy-acetone phosphate under standard assay conditions.

SDS-PAGE analysis of NADP⁺-dependent G3PDH from A. fulgidus heterologously produced in E. coli. About 5 μg protein were loaded on a 12% gel. After electrophoresis, the gels were stained with Coomassie Blue R250. (Lane 0) molecular mass standard; (lane 1) cell extract; (lane 2) supernatant after heat treatment; (lane 3) G3PDH fraction after chromatography on Resource Q; (lane 4) G3PDH fraction after chromatography on Hydroxyapatite; (lane 5) G3PDH fraction after chromatography on Source 15 Phe.

Effect of temperature on NADP⁺-dependent G3PDH activity of A. fulgidus. (A) Activity vs. temperature (°C). (B) Arrhenius plot of the same data. The activity was determined by following NADPH oxidation with dihydroxyacetone phosphate at pH 6.6 in 50 mM potassium phosphate buffer at the temperatures indicated. From the slopes of the linear parts in the Arrhenius plot an activation energy of 51 kJ/mol was calculated for the temperature range between 50°C and 75°C and of 22 kJ/mol for the temperature range between 30°C and 45°C.

Superposition (A) of the NADP⁺-dependent G3PDH monomer from A. fulgidus (blue) and of the G3PDH monomer from L. mexicana (green). The overall r.m.s.d. value between these models was 2.0 Å for 314 structurally equivalent residues. The loop between the indicated α-helices α14 and α15 is visible in the structure of G3PDH from A. fulgidus but is disordered in the structure of G3PDH from L. mexicana, the only other structurally solved G3PDH (), if the substrate binding site is not fully occupied. The substrates NADPH and dihydroxyacetone phosphate were modeled into the structure of the A. fulgidus G3PDH at the corresponding binding sites known from the L. mexicana G3PDH structure (). The structures of the L. mexicana enzyme and of the enzyme substrate complex were taken from the RCSB Protein Data Bank (accession codes 1N1E and 1EVY). (B) Active site region of A. fulgidus G3PDH showing the predicted binding modes of the substrates glycerol-3-phosphate and NADP⁺. The substrate molecules were modeled into the structure using the SCULPT option of the program PyMOL, which permits interactive energy minimization.