Presence and role of a second disulphide bond in recombinant lupanine hydroxylase using site-directed mutagenesis with ~(143)Cys→Ser and ~(124,143)Cys→Ser mutations in Escherichia coli

摘要

Lupanine hydroxylase (LH), a quinohaemoprotein, catabolizes lupanine and possesses four cysteine (Cys) residues; two associated with a cytochrome c motif (~(586)Cys and ~(589)Cys), while the role of the remaining two residues (~(124)Cys and ~(143)Cys) is unclear. Structural graphic simulation using homology modelling suggested a potential second -S-S- bond, a common feature between adjacent Cys residues in other quinohaemoproteins; however, in LH, these residues are located 18 amino acids apart. Formation of the second disulphide bond was initially chemically confirmed by iodomethane alkylation with 91% loss of enzymic activity, and no significant change was observed with unreduced alkylated protein. Dithiothreitol-induced reduction of LH followed by Cd ~(2+) treatment also resulted in significant loss of activity in a dose-dependent manner. Subsequent investigation into the role of disulphide bond in LH was performed using engineered ~(143)Cys→Ser and ~(124,143)Cys→Ser mutants and exhibited 25% and zero activity, respectively, of wild type in the periplasm. Homology structure prediction showed three changes in α-helices and four in β-pleated sheets in ~(143)Cys→Ser mutant, and ~(124,143)Cys→Ser mutant had six changes in α-helices and nine in β-pleated sheets. These mutations resulted in the enlargement of the molecule and affect the enzyme activity because of structural changes in the cytochrome c domain.