ATP-NADH kinase phosphorylates NADH to produce NADPH at the expense of ATP. The present study describes Fusarium oxysporum NADH kinase (ATP:NADH 2'-phosphotransferase, EC 2.7.1.86), a novel fungal enzyme capable of synthesizing NADPH using NADH as the preferred diphosphonicotinamide (diphosphopyridine) nucleotide donor. NADH kinase was highly purified (similar to 66-fold) and the enzyme was found to be a homodimeric with a subunit of M-r 72,000. Isoelectric focusing in the pH range of 3.0-9.5 of the purified NADH kinase yielded a pl value of about 5.6. The K-m values of NADH kinase for NADH and ATP were found to be 0.13 and 2.59 mM, respectively. Prediction of the secondary structure of the protein was performed in the PSIPRED server while modelling the three-dimensional (3D) structure was accomplished by the use of the HH 3D-structure prediction server. (C) 2008 Elsevier Ltd. All rights reserved.