Abstract
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.
Highlights
Lysyl hydroxylase (EC 1.14.11.4), an ␣2 homodimer, catalyzes the formation of hydroxylysine in collagens
Data on the four asparagine mutations suggested that only two of the potential Nglycosylation sites may be fully glycosylated in insect cells and that one of these carbohydrate units may be needed for full enzyme activity
The critical role of hydroxylysine is demonstrated by the marked changes in the mechanical properties of certain tissues seen in patients with the type VI variant of the Ehlers-Danlos syndrome [3,4,5], which is caused by mutations in the gene for lysyl hydroxylase (6 –10)
Summary
Lysyl hydroxylase (EC 1.14.11.4), an ␣2 homodimer, catalyzes the formation of hydroxylysine in collagens. 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. Data on the four asparagine mutations suggested that only two of the potential Nglycosylation sites may be fully glycosylated in insect cells and that one of these carbohydrate units may be needed for full enzyme activity. The hydroxylysine residues have two important functions Their hydroxy groups act as attachment sites for carbohydrate units, and they are essential for the stability of the intermolecular collagen crosslinks The O2 is thought to be bound end-on in an axial position, and the subsequent decarboxylation of 2-oxoglutarate is assumed to lead to the formation of a ferryl ion, which hydroxylates a lysine residue [1, 16]
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