AarF domain containing kinase 3 (ADCK3) is a mitochondrial protein known to have a role in the electron transport chain. Despite being required for the biosynthesis of coenzyme Q(10), a lipid-soluble electron transporter found to be essential for aerobic cellular respiration, the precise biological function of ADCK3 remains unknown. Patients with mutations in ADCK3 experience an onset of neurological disorders from childhood, including cerebellar ataxia and exercise intolerance. After extensive screening for soluble recombinant protein expression, an N-terminal fusion of maltose-binding protein was found to facilitate the overexpression of the human ADCK3 kinase domain in Escherichia coli as a soluble and biologically active entity. For the first time our work reveals Mg2+-dependent ATPase activity of ADCK3, providing strong support for the theoretical prediction of this protein being a functional atypical kinase. (C) 2014 Elsevier Inc. All rights reserved.