Human lysyl hydroxylase : identification of the residue involved in the binding of 2-oxoglutarate at the catalytic site and characterization of a novel isoenzyme, LH3, and its gene
|Organizations:||University of Oulu, Collagen Research Unit
University of Oulu, Biocenter Oulu
University of Oulu, Faculty of Medicine, Department of Medical Biochemistry and Molecular Biology
|Online Access:||PDF Full Text (PDF, 1 MB)|
|Persistent link:|| http://urn.fi/urn:isbn:9514257324
Oulu : University of Oulu,
|Publish Date:|| 2000-08-15
|Thesis type:||Doctoral Dissertation
|Defence Note:||Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium of the Department of Medical Biochemistry, on September 14th, 2000, at 10 a.m.
Professor Juhani Syväoja
Professor Heather N. Yeowell
Lysyl hydroxylase (E.C. 18.104.22.168, protocollagen-lysine 2-oxoglutarate 5-dioxygenase, PLOD) catalyses the formation of hydroxylysine in collagens and other proteins with collagen-like sequences. The hydroxylysine residues participate in the formation of collagen crosslinks and serve as attachment sites for carbohydrate units. The importance of lysine hydroxylation is demonstrated by the critical manifestations found in patients with the type VI variant of the Ehlers-Danlos syndrome, which is caused by a deficiency in lysyl hydroxylase activity.
Lysyl hydroxylase requires Fe2+, 2-oxoglutarate, O2 and ascorbate. The binding site for the C-5 carboxyl group of 2-oxoglutarate is characterized here by site-directed mutagenesis. Two conserved and one non-conserved amino acid residues at the possible binding site in human lysyl hydroxylase 1 were converted individually to alanine or lysine and the mutant polypeptides were expressed in insect cells. Mutation of arginine-700 to alanine inactivated the enzyme completely, whereas mutation of the other two residues had only a minor effect. In addition, the Km of the arginine-700 to lysine mutant polypeptide for 2-oxoglutarate was increased 10-fold. The results thus indicate that this conserved arginine is the residue that binds the C-5 carboxyl group of 2-oxoglutarate in lysyl hydroxylases.
A novel human lysyl hydroxylase isoenzyme, termed lysyl hydroxylase 3, was identified, cloned and characterized here. The overall amino acid sequence identity between the novel human lysyl hydroxylase isoenzyme and the other human lysyl hydroxylase isoenzymes is about 60%. The highest expression levels of the mRNA for lysyl hydroxylase 3 among the tissues studied were found in the placenta, pancreas and heart. The novel isoenzyme was expressed as a recombinant protein in insect cells, and the protein was shown to function as a lysyl hydroxylase in vitro hydroxylation experiments using short synthetic peptides as substrates. No differences in catalytic properties were found between the recombinant lysyl hydroxylases 3 and 1.
The structure of the human gene for lysyl hydroxylase 3 was determined in the last part of this work. The gene is shown to be only 11.6 kb in size and to contain 19 exons. Transcription was found to be initiated at multiple sites, and the introns contained 15 full-length Alu retroposons or partial Alu fragments of more than 100 bp. The present characterization of the exon-intron organization of the gene will provide a basis for further studies to determine whether there is any genetic disease that is attributable to mutations in this gene.
Acta Universitatis Ouluensis. D, Medica
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