Effect of active-site aromatic residues Tyr or Phe on activity and stability of glucose 6-phosphate dehydrogenase from psychrophilic Arctic bacterium Sphingomonas sp

Cold-adapted enzymes maintain correct conformation at their active sites despite their intrinsically flexible structures. The psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621 has two glucose 6-phosphate dehydrogenase (G6PD) isozymes, SpG6PD1 involved in the Entner-Doudoroff pathway and SpG6PD2 in the oxidative pentose phosphate pathway. Structural modeling of SpG6PD1 showed that the hydroxyl group of Tyr(177) participates in substrate binding by forming a hydrogen bond with the phosphate group of glucose 6-phosphate, whereas in SpG6PD2, a Phe residue is present in the corresponding position of Tyr(177). In this study, we investigated how subtle differences in aromatic residues in the substrate-binding pocket of SpG6PD1 affect enzymatic activity and stability. Mutations of Tyr(177) to Ala, His, Phe, and Trp caused increases in the rigidity of the SpG6PD1 structure. Particularly, mutants Y177F and Y177W showed increased thermal stabilities compared to wild-type (WT) but 3and 15-fold lower catalytic efficiencies, respectively. However, mutants Y177A and Y177H became heat-labile at moderate temperatures. These results indicate that an aromatic residue (Tyr or Phe) is necessary for the substrate-binding pocket of SpG6PD1; Tyr with its hydroxyl group is preferred for enzymatic activity, whereas the more hydrophobic Phe is preferred for thermal stability. Substitutions of bulky Trp for Tyr or Phe at this position resulted in substantial loss of activity. Our study suggests that delicate adjustment of aromatic residues can regulate the activity and stability of psychrophilic G6PD isozymes involved in different metabolic pathways.

Automated summary

This study investigates the impact of subtle differences in aromatic residues in the substrate-binding pocket of psychrophilic G6PD isozymes on enzymatic activity and stability. Results show that an aromatic residue (Tyr or Phe) is crucial for substrate binding in SpG6PD1, with Tyr preferred for enzymatic activity and Phe preferred for thermal stability. Substitution of bulky Trp for Tyr or Phe leads to significant loss of activity.