Biochemical comparison of two glucose 6-phosphate dehydrogenase isozymes from a cold-adapted Pseudomonas mandelii

Cold-adapted bacteria primarily have two glucose 6-phosphate dehydrogenase isozymes (G6PD, also known as zwf), zwf-1 for the Entner–Doudoroff pathway and zwf-2 for the oxidative pentose phosphate pathway. Although the roles of zwfs in carbon metabolism and antioxidant defense have been reported, the biochemical properties of zwfs at low and moderate temperatures have not been fully described. In this study, we cloned and characterized zwf-1 (Pmzwf-1) and zwf-2 (Pmzwf-2) from a cold-adapted bacterium Pseudomonas mandelii JR-1. Pmzwf-1 and Pmzwf-2 were expressed in Escherichia coli BL21 (DE3) as soluble tetrameric proteins. Both Pmzwf proteins were active at 4 °C, but Pmzwf-1 exhibited overall better biochemical properties than those of Pmzwf-2, including 10–30% higher specific activity at 4–40 °C as well as consistent conformational flexibility and thermal stability in the 4–40 °C range. Pmzwf-2 showed reduced thermal stability at moderate temperatures. Furthermore, the mRNA expression of Pmzwf-1 was higher than that of Pmzwf-2 at both 4 °C and 25 °C. These results indicate that Pmzwfs are cold-adapted enzymes, but Pmzwf-1 can function at both low to moderate temperatures while Pmzwf-2 is primarily functional at low temperatures. Our results suggest distinct temperature adaptations of two G6PD isozymes in P. mandelii JR-1, adaptations that are metabolic pathway dependent.