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

Biochim Biophys Acta Proteins Proteom. 2020 Feb;1868(2):140331. doi: 10.1016/j.bbapap.2019.140331. Epub 2019 Nov 22.

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 a different mechanism stabilizes the enzyme from Pseudomonas aeruginosa (PaG6PDH) (EC 1.1.1.363). The spectrofluorometric analysis of the emission of 8-anilino-1-naphthalenesulfonic acid (ANS) indicates that this stabilization is the result of a structural change in the enzyme caused by G6P. The similarity between the Kd values determined for the PaG6PDH-G6P complex (78.0 ± 7.9 μM) and the K0.5 values determined for G6P (57.9 ± 2.5 and 104.5 ± 9.3 μM in the NADP+- and NAD+-dependent reactions, respectively) suggests that the structural changes are the result of G6P binding to the active site of PaG6PDH. Modeling of PaG6PDH indicated the residues that potentially bind the ligand. These results and a phylogenetic analysis of the amino acid sequences of forty-four G6PDHs, suggest that the stabilization observed for PaG6PDH could be a characteristic that distinguishes this and other G6PDHs that use NAD+ and NADP+ from those that use NADP+ only or preferentially, such as those found in eukaryotes. This characteristic could be related to the metabolic roles these enzymes play in the organisms to which they belong.

Keywords: Conformational changes; Entner-Doudoroff pathway; Glucose-6-phosphate dehydrogenase; Pseudomonas aeruginosa; Structural stability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Anilino Naphthalenesulfonates / chemistry
  • Binding Sites
  • Catalytic Domain
  • Glucose-6-Phosphate / chemistry
  • Glucose-6-Phosphate / metabolism
  • Glucosephosphate Dehydrogenase / classification
  • Glucosephosphate Dehydrogenase / genetics
  • Glucosephosphate Dehydrogenase / metabolism*
  • Kinetics
  • Molecular Dynamics Simulation
  • NAD / metabolism
  • NADP / chemistry
  • NADP / metabolism
  • Phylogeny
  • Protein Binding
  • Protein Denaturation
  • Protein Stability
  • Protein Structure, Tertiary
  • Pseudomonas aeruginosa / enzymology*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification

Substances

  • 8-anilino-1-naphthalenesulfonic acid
  • Anilino Naphthalenesulfonates
  • Recombinant Proteins
  • NAD
  • NADP
  • Glucose-6-Phosphate
  • Glucosephosphate Dehydrogenase