Lysine: N 6 -Hydroxylase: Stability and Interaction with Ligands

Abstract

Recombinant lysine:N 6 -hydroxylase, r IucD, which is isolated as an apoenzyme, requires FAD and NADPH for its catalytic function. r IucD preparations have been found to undergo time-dependent loss in monooxygenase function due to aggregation from the initial tetrameric state to a polytetrameric form(s), a process which is reversible by treatment with thiols. Ligand-in-duced conformational changes in r IucD were assessed by monitoring its CD spectra, DSC profile, and susceptibility to both endo- as well as exopeptidases. The first two methods indicated the absence of any significant conformational change in r IucD, while the last approach revealed that FAD, and its analog ADP, can protect the protein from the deleterious action of proteases. NADPH was partially effective and L-lysine was ineffective in this regard. Deletion of the C-terminal segment, either by treatment with carboxypeptidase Y or by mutagenesis of iucD, results in the loss of r IucD's monooxygenase activity. These findings demonstrate the crucial role of the C-terminal segment in maintaining r IucD in its native conformation.