4-Hydroxy benzaldehyde dehydrogenase catalyses a step in a well-studied pathway for the catabolism of 4-hydroxy-substituted cinnamates in Acinetobacter baylyi, oxidising a benzaldehyde group to the corresponding benzoic acid, with the concomitant reduction of NAD(+) to NADH. Although much genetic and in vivo data for the enzyme have been presented, there have been no reports of the purification and in vitro characterisation of this enzyme. In this work, 4-hydroxy benzaldehyde dehydrogenase from A. baylyi was cloned to incorporate a hexahistidine affinity tag, heterologously expressed in Escherichia coli, purified and characterised. The enzyme was found to be stable up to 45 degrees C, losing only 25% of its activity over 9 h at this temperature. NAD(+) was the preferred cofactor. The enzyme was found to act on a number of benzaldehyde substrates, and steady-state kinetics suggested that 4-hydroxy and 3,4-dihydroxy benzaldehydes were preferred substrates to benzaldehyde, or 4-hydroxy 3-methoxy benzaldehyde (vanillin). (C) 2008 Elsevier Inc. All rights reserved.