Presence and role of a second disulphide bond in recombinant lupanine hydroxylase using site-directed mutagenesis with

Author： Stampolidis Pavlos Kaderbhai Naheed N. Kaderbhai Mustak A.

ISSN： 0378-1097

Source： FEMS Microbiology Letters, Vol.334, Iss.1, 2012-09, pp. : 35-43

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Abstract

AbstractLupanine hydroxylase (LH), a quinohaemoprotein, catabolizes lupanine and possesses four cysteine (Cys) residues; two associated with a cytochrome c motif (⁵⁸⁶Cys and ⁵⁸⁹Cys), while the role of the remaining two residues (¹²⁴Cys and ¹⁴³Cys) 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 Cd²⁺ 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 ¹⁴³Cys→Ser and ^124,143Cys→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 ¹⁴³Cys→Ser mutant, and ^124,143Cys→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.