Site-directed mutagenesis of human lysyl hydroxylase expressed in insect cells. Identification of histidine residues and an aspartic acid residue critical for catalytic activity.
Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 homodimer, catalyzes the formation of hydroxylysine in collagens. We expressed here human lysyl hydroxylase in insect cells by baculovirus vectors. About 90% of the enzyme produced was soluble 32 h after infection, whereas only 10% was soluble at 72 h. Twelve histidines, five aspartates, and all four asparagines that may act as N-glycosylation sites were converted individually to serine, alanine, or glutamine, respectively, and the mutant enzymes were expressed in insect cells. Three histidine mutations and one aspartate mutation appeared to inactivate the enzyme completely. These and other data suggest that histidines 656 and 708 and aspartate 658 provide the three ligands required for the binding of Fe2+ to a catalytic site, whereas the role of the third critical histidine (residue 706) remains to be established. Three additional histidine mutations also had a major effect, although they did not inactivate the enzyme completely, whereas six further histidine mutations and four out of five aspartate mutations had a much more minor effect. Data on the four asparagine mutations suggested that only two of the potential N-glycosylation sites may be fully glycosylated in insect cells and that one of these carbohydrate units may be needed for full enzyme activity.
Pirskanen
A
A
Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Finland.
Kaimio
A M
AM
Myllylä
R
R
Kivirikko
K I
KI
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D001224:Aspartic Acid; D016367:Baculoviridae; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006639:Histidine; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D003062:Codon; D011994:Recombinant Proteins; D001224:Aspartic Acid; D006639:Histidine; D001216:Asparagine; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase