Pentose phosphate pathway (PPP) is a metabolic pathway parallel to glycolysis, which generates nicotinamide adenine dinucleotide phosphate (NADPH) for reductive biosynthesis reactions and pentoses as well as such as ribose 5-phosphate, a precursor for the synthesis of nucleotides. 6-phosphogluconate is an intermediate in PPP which metabolize glucose 6-phosphate to glyceraldehyde 3-phosphate. In bacteria, it is known that alternative pathway of PPP metabolize glucose to 6-phosphogluconate. However, it has not been reported in human and mouse. To reveal a novel alternative PPP, we identified gluconokinase (GNK) gene and characterized GNK activity for phosphorylating gluconte to 6-phosphogluconate.

By NCBI-BLAST search, amino acid sequence of bacterial GNK in Pseudomonas putida KT2440 showed high homology with those of possible GNK gene (NM_001001551 and NM_198004) in human and mouse, respectively. We cloned cDNA of possible GNK gene from liver in human and mouse. Recombinant 6xHis-tagged proteins were purified from bacterial soluble fraction by using Ni-NTA agarose and DEAE-Sephacel column. GNK activity in reaction mixture containng gluconate, ATP, MgCl₂, and recombinant GNK protein was measured by monitoring A₃₄₀ of NADPH generated in coupled with resulting 6-phosphogluconate, NADP⁺, and 6-phosphogluconate dehydrogenase. Human and mouse recombinat proteins showed GNK activity in dose-dependent manner, respectively. Furthermore, GNK activity was detected in mouse liver extract.

In this study, we identified GNK gene in human and mouse, suggesting that alternative PPP could exists in human and mouse.