Yarrowia lipolytica, a nonpathogenic, nonconventional, aerobic and dimorphic yeast, is considered an oleaginous microorganism due to its excellent ability to accumulate large amounts of lipids. Glucose-6-phosphate dehydrogenase (G6PD) is one of two key enzymes involved in the lipid accumulation in this fungi, which catalyzes the oxidative dehydrogenation of glucose-6-phosphate to 6-phosphoglucono-delta-lactone with the reduction of NADP (+) to NADPH. In this study, the full-length gene of G6PD from Y. lipolytica (YIG6PD) was cloned without intron and heterogeneously expressed in E. coli Then, YIG6PD was purified and biochemically characterized in details. Kinetic analysis showed that YIG6PD was completely dependent on NADP(+) and its apparent K-m for NADP(+) was 33.3 mu M. The optimal pH was 8.5 and the maximum activity was around 47.5 degrees C. Heat-inactivation profiles revealed that it remained 50% of maximal activity after incubation at 48 degrees C for 20 min YIG6PD activity was competitively inhibited by NADPH with a K-i value of 56.04 mu M. Most of the metal ions have no effect on activity, but Zn2+ was a strong inhibitor. Furthermore, the determinants in the coenzyme specificity of YIG6PD were investigated. Kinetic analysis showed that the single mutant R52D completely lost the ability to utilize NADP(+) as its coenzyme, suggesting that Arg-52 plays a decisive role in NADP (+) binding in YIG6PD. The identification of Y. lipolytica G6PD may provide useful scientific information for metabolic engineering of this yeast as a model for bio-oil production.