The substrate of the glucose-6-phosphate dehydrogenase of pseudomonas aeruginosa provides structural stability

Abstract

In general, eukaryotic glucose-6-phosphate dehydrogenases (G6PDHs) are structurally stabilized by NADP+.Here we show by spectrofluorometric analysis, thermal and urea denaturation, and trypsin proteolysis, that adifferent mechanism stabilizes the enzyme fromPseudomonas aeruginosa(PaG6PDH) (EC 1.1.1.363). The spec-trofluorometric analysis of the emission of 8-anilino-1-naphthalenesulfonic acid (ANS) indicates that this sta-bilization is the result of a structural change in the enzyme caused by G6P. The similarity between theKdvaluesdetermined for the PaG6PDH-G6P complex (78.0 ± 7.9μM) and the K0.5values determined for G6P(57.9 ± 2.5 and 104.5 ± 9.3μM in the NADP+- and NAD+-dependent reactions, respectively) suggests thatthe structural changes are the result of G6P binding to the active site of PaG6PDH. Modeling of PaG6PDHindicated the residues that potentially bind the ligand. These results and a phylogenetic analysis of the aminoacid sequences of forty-four G6PDHs, suggest that the stabilization observed for PaG6PDH could be a char-acteristic that distinguishes this and other G6PDHs that use NAD+and NADP+from those that use NADP+onlyor preferentially, such as those found in eukaryotes. This characteristic could be related to the metabolic rolesthese enzymes play in the organisms to which they belong.