Presence and role of a second disulphide bond in recombinant lupanine hydroxylase using site-directed mutagenesis with 143Cys→Ser and 124,143Cys→Ser mutations in Escherichia coli.
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.
Department of Molecular Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
Pavlos
P
Stampolidis
Naheed N
NN
Kaderbhai
Mustak A
MA
Kaderbhai
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011549:Pseudomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D004220:Disulfides; C023929:lupanine hydroxylase; D000587:Oxidoreductases Acting on CH-NH Group Donors