Structure of human aldehyde oxidase under tris(2-carboxyethyl)phosphine reducing conditions.

The importance of human aldehyde oxidase (hAOX1) has increased in recent decades due to its involvement in drug metabolism. Inhibition studies involving hAOX1 are extensive and a common reducing agent, dithiothreitol (DTT), was recently found to inactivate the enzyme. However, in previous crystallographic studies of hAOX1, DTT was found to be essential for crystallization. To surpass this concern, another reducing agent was used in crystallization trials. Using tris(2-carboxyethyl)phosphine (TCEP), a sulfur-free reducing agent, it was possible to obtain well ordered crystals of wild-type hAOX1 and a variant, hAOX1_6A, which diffracted beyond 2.3 Å resolution. Instead of the typical star-shaped crystals of hAOX1, at pH 4.7 plates are obtained in the orthorhombic space group P21212 with two molecules in the asymmetric unit. Activity assays with the enzyme incubated with both reducing agents show that in contrast to DTT, TCEP did not inactivate hAOX1. The replacement of DTT with TCEP in the crystallization of hAOX1 provides a strategy to circumvent enzyme inactivation during crystallographic studies, allowing future applications of new assays, such as time-resolved crystallography.