Recombinant lysine:N§6-hydroxylase, conformational response to Cys --> Ala substitutions and interaction with its ligands

Abstract

Recombinant Lysine:N"-hydroxylase (rlucD) catalyses the conversion of L-lysine to its N 6 -hydroxy derivative and requires flavin adenine dinucleotide (FAD) and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) for its catalytic function. Previous studies had revealed that, of the five cysteine residues present in riucD, two of them were susceptible co alkylation with iodoacetate and three such residues were amenable to modification by S,5'-dithiobis-(2-nitrobenzoic acid), DTNB, with the concomitant loss of lysine monooxygenase function. Furthermore, t\.VO of riucD's alkylatable cysteine residues, CysSl and Cys158, could be replaced, by alanine, without adverse effect on the catalytic function (L Marrone and T. Viswanatha, BiochinL Biopf!Js. Acta 1343, 263-277, 1997).

In the current investigations, the effect of individual replacement of rlucD's Cys31, Cysl 46, and Cys 166 with alanine on the protein's structural integrity and catalytic function was examined by monitoring T11., using differential scanning calorimetry and lysine:Nr,_ hydroxylase activity respectively. Each of these substitutions appeared to lead to a slight decrease in the stability of the protein, as indicated by a lower TM relative to that of the parent protein. Interestingly, the Cys166-tAla substitution, which was found to be accompanied by the largest decrease in TM (-2.4 K), had no significant effect on the protein's catalytic function. In contrast, Cysl46-tAla replacement, which results in a relatively much 10\ver decrease in T"' (-0.8 I<), was accompanied by profound conformational changes as indicated by approximately 50% reduction in monooxygenase activity and the ready accessibility of the remaining cysteine residues to chemical modification.

Ligand-induced conformational changes in rlucD 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 rlucD, 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 i11cD, results in the loss of rlucD's monooxygenase activity. These findings demonstrate the crucial role of the C-terminal segment in maintaining rlucD in its native conformation.