Prolyl 4-hydroxylase : structural and functional characterization of the peptide-substrate-binding domain of the human enzyme, and cloning and characterization of a plant enzyme with unique properties
1University of Oulu, Collagen Research Unit
2University of Oulu, Biocenter Oulu
3University of Oulu, Faculty of Medicine, Department of Medical Biochemistry and Molecular Biology
|Online Access:||PDF Full Text (PDF, 0.8 MB)|
|Persistent link:|| http://urn.fi/urn:isbn:9514271793
|Publish Date:|| 2003-10-24
|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 L101 of the Department of Medical Biochemistry and Molecular Biology, on October 24th, 2003, at 10 a.m.
Professor Neil J. Bulleid
Docent Volkmar Günzler
Collagen prolyl 4-hydroxylase is the key enzyme in the biosynthesis of collagens, a family of extracellular matrix proteins. Vertebrate collagen prolyl 4-hydroxylases are α₂β₂ tetramers, the β subunit being identical to the multifunctional protein disulphide isomerase (PDI). Several isoforms of the catalytic α subunit have been identified in various organisms. Prolyl 4-hydroxylases have also been isolated from plants, where they hydroxylate proline-rich structural glycoproteins of the cell walls.
The structural and functional properties of the peptide-substrate-binding domain of human collagen prolyl 4-hydroxylase are characterized here. Data obtained from NMR studies indicate that the domain consists of five α helices and one short β strand, this structure being quite different from those of other proline-rich peptide-binding modules. Several residues involved in the binding of a short synthetic peptide were also identified by NMR. Kd values for the binding of several synthetic peptides to the α(I) and α(II) domains were determined by surface plasmon resonance and isothermal calorimetry, and the results indicated that the binding properties of the type I and type II collagen prolyl 4-hydroxylase tetramers can mainly be explained by the binding of peptides to this domain rather than to the catalytic domain.
The peptide-substrate-binding domain of human type I collagen prolyl 4-hydroxylase was also crystallized. The crystals were well ordered and diffracted to at least 3 Å, the asymmetric unit most probably containing a domain dimer.
The genome of Arabidopsis thaliana was found to encode at least six putative prolyl 4-hydroxylase polypeptides, one of which was cloned and characterized here as a recombinant protein. All the catalytically critical residues identified in animal prolyl 4-hydroxylases were also conserved in this plant prolyl 4-hydroxylase, and their mutagenesis led to inactivation of the enzyme. The recombinant plant enzyme was effective in hydroxylating poly(L-proline) and several synthetic proline-rich peptides. Surprisingly, contrary to previous reports on plant prolyl 4-hydroxylases, the collagen-like peptides were found to be good substrates, the enzyme preferentially hydroxylating prolines in the Y positions of the -X-Y-Gly- triplets, thus resembling the vertebrate collagen prolyl 4-hydroxylases even in this respect. The recombinant plant prolyl 4-hydroxylase also hydroxylated peptides representing the N and C-terminal hydroxylation sites present in the hypoxia-inducible transcription factor α. The fact that these peptides contain only one proline residue indicated that a poly(L-proline) type II conformation was not required for hydroxylation.
Acta Universitatis Ouluensis. D, Medica
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