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 Tyr¹⁷⁷ 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 Tyr¹⁷⁷. 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 Tyr¹⁷⁷ 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 3- and 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.

尽管它们具有固有的柔性结构，但经过冷适应的酶在其活性位点仍保持正确的构象。嗜冷的北极细菌鞘氨醇单胞菌。PAMC 26621具有两个6-葡萄糖葡萄糖脱氢酶（G6PD）同工酶，参与Entner-Doudoroff途径的SpG6PD1和参与氧化戊糖的途径的SpG6PD2。SpG6PD1的结构模型表明，Tyr ¹⁷⁷的羟基通过与6-磷酸葡萄糖的磷酸基形成氢键参与底物结合，而在SpG6PD2中，Phe残基存在于Tyr ¹⁷⁷的相应位置。在这项研究中，我们调查了SpG6PD1的底物结合口袋中的芳香族残基的细微差异如何影响酶的活性和稳定性。Tyr ^177的变异对Ala，His，Phe和Trp的影响导致SpG6PD1结构的刚度增加。特别地，与野生型（WT）相比，突变体Y177F和Y177W显示出增加的热稳定性，但是催化效率分别降低了3和15倍。但是，突变体Y177A和Y177H在中等温度下变得不耐热。这些结果表明，芳香族残基（Tyr或Phe）对于SpG6PD1的底物结合口袋是必不可少的。具有其羟基的Tyr对于酶促活性是优选的，而更疏水的Phe对于热稳定性是优选的。在该位置用大体积的Trp替代Tyr或Phe导致活性大大降低。我们的研究表明，芳香族残基的精细调节可以调节参与不同代谢途径的嗜酸性G6PD同工酶的活性和稳定性。