A Cys/Ser mutation of NDPK-A stabilizes its oligomerization state and enhances its activity

Abstract

Nucleoside diphosphate phosphate transferase A (NDPK-A) is a multifunction protein encoded by the metastasis suppressor gene nm23-H1 that can catalyse the transfer of a phosphoryl group from nucleoside triphosphates to nucleoside diphosphates to maintain the pool of nucleoside triphosphate required for biosynthesis (1, 2). It is involved in cellular regulatory functions such as cell proliferation, differentiation, development and apoptosis. However, the regulation and mechanism of the multifunctions of NDPK are poorly understood, especially with respect to oxidative modification. It has been proposed that oxidative modification is a possible means of regulating the functions of NDPK in many cellular processes, based on in vitro experimental results (3). The activity of nucleoside diphosphate (NDP) transferase in human brain is decreased in Alzheimer’s disease and Down syndrome, and this might result from the oxidative modification of NDPK (4). In a previous study, we found that the NDP transferase activity of NDPK increased 2-fold after treatment with N-acetylcysteine, and treatment with H₂O₂ resulted in inactivation of the enzyme for the formation of intermolecular disulphide bonds (DBs) and disruption of its oligomerization state (5). NDPK-A is catalytically active in its hexameric form and inactive in its dimeric form (6–8). It has been reported that the native hexameric structure is dissociated into a dimeric form by the intermolecular DBs of NDPK (3). There are three cysteine residues in NDPK-A, i.e. Cys4, Cys109 and Cys145. In previous peptide mapping study, we demonstrated that Cys4 and Cys145 form a DB in the oxidized state of NDPK-A (5). Here, we have mutated the cysteine 145 (C145) residue of NDPK-A to serine using site-directed mutagenesis, and the physicochemical characteristics and bioactivities of NDPK-A C145S were determined to investigate the possible role of DB isomerization between C145 and the other cysteine residues.