Heteroexpression and biochemical characterization of a glucose-6-phosphate dehydrogenase from oleaginous yeast Yarrowia lipolytica

Heteroexpression and functional characterization of glucose 6-phosphate dehydrogenase from industrial Aspergillus oryzae

“…The optimal temperature was around 50°C (Fig. 3B), which was similar to the known G6PDHs (30-92°C) [21]. The G6PDH had an approximately 60-80% of its maximum activity at 35-45°C, but only ~2 and ~30% of its maximum activity at 60°C and 20°C were gauged, respectively.…”

“…A motif (RXXXEKPXG) in the coenzyme-binding site and three conserved motifs (annotation) (RIDHYLGK, EXXGXEXRXXY and DXXQNH) in the substrate-binding site were also observed in AoG6PDH (Fig. S2), indicating the G6P and NADP + binding and catalyzing residues were highly conserved [21]. The secondary structure of the protein was analyzed, the α-helix region occupied 46.7%, the β-sheet was 12.8% and the random coil was 40.6% (Fig.…”

“…According to the specificity of coenzyme, G6PDH can be divided into three categories: NADP [27], A. nidulans [27], A. parasiticus [28] and A. aculeatus [29], also exhibited strict specificity towards NADP + . In all NADP + preferred G6PDHs, the conserved Arg residue was revealed and identified as a determinant in the NADP + specificity [21]. The conserved Arg residue binds tightly with the negatively charged phosphate group of NADP + via forming an electrostatic interaction [21].…”

“…In all NADP + preferred G6PDHs, the conserved Arg residue was revealed and identified as a determinant in the NADP + specificity [21]. The conserved Arg residue binds tightly with the negatively charged phosphate group of NADP + via forming an electrostatic interaction [21]. The corresponding Arg residue was assessed in AoG6PDH, while, in some dual coenzyme specific enzymes the corresponding Arg residue still existed [30].…”

“…The activity of G6PDH was determined using the methods described with modifications [21]. G6PDH activities were measured by monitoring NADPH formation at 340 nm and at 25°C on a spectrophotometer, equipped with a thermo-stated cuvette holder.…”