Collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of 4-hydroxyproline by the hydroxylation of -X-Pro-Gly-triplets. The vertebrate enzymes are ␣ 2 ␤ 2 tetramers, the ␤-subunit being identical to protein-disulfide isomerase (PDI). Two isoforms of the catalytic ␣-subunit, which combine with PDI to form [␣(I)] 2 ␤ 2 and [␣(II)] 2 ␤ 2 tetramers, have been known up to now. We report here on the cloning and characterization of a third vertebrate C-P4H ␣-subunit isoform, ␣(III). The processed human, rat and mouse ␣(III) polypeptides consist of 520 -525 residues, all three having signal peptides of 19 -22 additional residues. The sequence of the processed human ␣(III) polypeptide is 35-37% identical to those of human ␣(I) and ␣(II), the highest identity being found within the catalytically important C-terminal region and all five critical residues at the cosubstrate binding sites being conserved. The sequence within a region corresponding to the peptide-substrate binding domain is less conserved, but all five ␣ helices constituting this domain can be predicted to be located in identical positions in ␣(I), ␣(II), and ␣(III) and to have essentially identical lengths. The ␣(III) mRNA is expressed in many human tissues, but at much lower levels than the ␣(I) and ␣(II) mRNAs. In contrast to ␣(I) and ␣(II), no evidence was found for alternative splicing of the ␣(III) transcripts. Coexpression of a recombinant human ␣(III) polypeptide with PDI in human embryonic kidney cells led to the formation of an active enzyme that hydroxylated collagen chains and a collagen-like peptide and appeared to be an [␣(III)] 2 ␤ 2 tetramer. The catalytic properties of the recombinant enzyme were very similar to those of the type I and II C-P4Hs, with the exception that its peptide binding properties were intermediate between those of the type I and type II enzymes.

The collagen prolyl 4-hydroxylases (P4Hs, EC 1.14.11.2) play a critical role in the synthesis of the extracellular matrix. The enzymes characterized from vertebrates and Drosophila are ␣ 2 ␤ 2 tetramers, in which protein disulfide isomerase (PDI) serves as the ␤ subunit. Two conserved ␣ subunit isoforms, PHY-1 and PHY-2, have been identified in Caenorhabditis elegans. We report here that three unique P4H forms are assembled from these polypeptides and the single ␤ subunit PDI-2, both in a recombinant expression system and in vivo, namely a PHY-1/PHY-2/(PDI-2) 2 mixed tetramer and PHY-1/PDI-2 and PHY-2/PDI-2 dimers. The mixed tetramer is the main P4H form in wild-type C. elegans but phy-2 ؊/؊ and phy-1 ؊/؊ (dpy-18) mutant nematodes can compensate for its absence by increasing the assembly of the PHY-1/PDI-2 and PHY-2/PDI-2 dimers, respectively. All three of the mixed tetramer-forming polypeptides PHY-1, PHY-2, and PDI-2 are coexpressed in the cuticle collagen-synthesizing hypodermal cells. The catalytic properties of the mixed tetramer are similar to those of other P4Hs, and analogues of 2-oxoglutarate were found to produce severe temperature-dependent effects on P4H mutant strains. Formation of the novel mixed tetramer was species-specific, and studies with hybrid recombinant PHY polypeptides showed that residues Gln 121 -Ala 271 and Asp 1 -Leu 122 in PHY-1 and PHY-2, respectively, are critical for its assembly.The free-living nematode Caenorhabditis elegans represents an excellent model system for studying the extracellular matrix (ECM) 1 and the enzymes involved in its biosynthesis and modification (1-3). The major ECM formed in C. elegans is the collagenous cuticle or exoskeleton, a protective structure that is synthesized repeatedly during development. Over 150 small collagen genes are involved in the formation of this structure (2), producing stage-specific cuticles that are both structurally and chemically distinct (3).Collagen prolyl 4-hydroxylases (P4Hs) are enzymes resident in the endoplasmic reticulum (ER) (4, 5) that play a critical role in the synthesis and processing of all collagens (6). Recently, an additional family of cytoplasmic P4Hs has been shown to play an essential role in O 2 sensing and the hypoxia response (7,8).The collagen P4Hs present in vertebrates (4, 5) and in Drosophila (9) are ␣ 2 ␤ 2 tetramers in which the multifunctional chaperone protein disulfide isomerase (PDI) serves as the ␤ subunit. The ␣ subunit binds Fe 2ϩ , 2-oxoglutarate, and ascorbate (4, 5) and possesses the peptide substrate-binding site (10). The main role of PDI is to retain the ␣ subunits in a catalytically active, nonaggregated conformation within the ER (11-13). Two conserved P4H ␣ subunit isoforms have been described in human and mouse tissues, which form [␣(I)] 2 ␤ 2 or [␣(II)] 2 ␤ 2 tetramers (14 -16). Initial characterization of a recombinant C. elegans ␣ subunit isoform (PHY-1) revealed a unique association in that it formed an active ␣␤ dimer with both human PDI and its C. elegans orthologue PDI-2 but no...

The collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of 4-hydroxyproline by the hydroxylation of proline residues in -Xaa-Pro-Gly-sequences. The vertebrate enzymes are ␣ 2 ␤ 2 tetramers in which protein-disulfide isomerase serves as the ␤ subunit. Two isoforms of the catalytic ␣ subunit have been identified and shown to form [␣(I)] 2 ␤ 2 and [␣(II)] 2 ␤ 2 tetramers, the type I and type II C-P4Hs, respectively. The peptide-substrate-binding domain of type I C-P4H has been shown to be located between residues 138 and 244 in the 517-residue ␣(I) subunit and to be distinct from the catalytic domain that is located in the C-terminal region. We report here that a recombinant human C-P4H ␣(I) polypeptide The prolyl 4-hydroxylases (P4Hs) 1 catalyze the formation of 4-hydroxyproline by the hydroxylation of proline residues in peptide linkages. Two P4H families are known today. The collagen P4Hs (C-P4Hs), enzymes residing within the lumen of the endoplasmic reticulum, have a central role in the synthesis of all collagens, the resulting 4-hydroxyproline residues being essential for assembly of the triple-helical molecules (1-3). The hypoxia-inducible factor (HIF) P4Hs, a family of cytoplasmic enzymes (4 -6), play a key role in the response of cells to hypoxia by catalyzing hydroxylation of the ␣ subunit of HIF. This subunit is synthesized continuously, and at least one of two critical proline residues becomes hydroxylated under normoxic conditions, the resulting 4-hydroxyproline being essential for rapid degradation of HIF-␣ (7-9). In hypoxia this hydroxylation ceases, HIF-␣ forms a dimer with HIF-␤, and the dimer then becomes bound to the HIF-responsive elements in a number of hypoxia-inducible genes.All vertebrate C-P4Hs are ␣ 2 ␤ 2 tetramers in which the ␤ subunit is identical to the enzyme and chaperone protein-disulfide isomerase (1-3). Two isoforms of the catalytic ␣ subunit have been characterized from human and mouse tissues and shown to form [␣(I)] 2 ␤ 2 and [␣(II)] 2 ␤ 2 tetramers, called the type I and type II C-P4Hs, respectively (10, 11). The type I enzyme is the most abundant form in most cells, but type II is the main form in chondrocytes, endothelial cells, and some other cell types (12, 13). The HIF-P4Hs appear to consist of only one type of monomer, the size of which ranges from 239 to 426 residues in the three human isoenzymes (4 -6).The C-P4Hs act on -Xaa-Pro-Gly-triplets in collagens and more than 15 other proteins with collagen-like sequences (1-3, 14), whereas the HIF-P4Hs hydroxylate -Leu-Xaa-Xaa-LeuAla-Pro-Tyr-and -Leu-Xaa-Xaa-Leu-Ala-Pro-Ala-sequences (7-9). All the P4Hs require Fe 2ϩ , 2-oxoglutarate, O 2 , and ascorbate, the 2-oxoglutarate being stoichiometrically decarboxylated during hydroxylation (1-3). The C-terminal regions of the C-P4H ␣ subunits and the HIF-P4H monomers contain four conserved residues, two histidines, and one aspartate that bind the Fe 2ϩ atom and a basic residue that binds the C-5 carboxyl group of the 2-oxoglutarate (15,16). This basic residue is a lysine ...

Winter, A. D. et al. (2007)

The collagen prolyl 4-hydroxylases (collagen P4Hs, EC 1.14.11.2) play a key role in the synthesis of the extracellular matrix. The vertebrate enzymes are ␣ 2 ␤ 2 tetramers, the ␤ subunit being identical to protein disulfide isomerase (PDI). The main Caenorhabditis elegans collagen P4H form is an unusual PHY-1/PHY-2/(PDI) 2 mixed tetramer consisting of two types of catalytic ␣ subunit, but the PHY-1 and PHY-2 polypeptides also form active PHY/PDI dimers. The lengths of peptide substrates have a major effect on their interaction with the P4H tetramers, the K m values decreasing markedly with increasing chain length. This phenomenon has been explained in terms of processive binding of the two catalytic subunits to long peptides. We determined here the K m values of a collagen P4H having two catalytic sites, the C. elegans mixed tetramer, and a form having only one such site, the PHY-1/PDI dimer, for peptides of varying lengths. All the K m values of the PHY-1/PDI dimer were found to be about 1.5-2.5 times those of the tetramer, but increasing peptide length led to identical decreases in the values of both enzyme forms. The K m for a nonhydroxylated collagen fragment with 33 -X-Y-Gly-triplets but only 11 -X-Pro-Gly-triplets was found to correspond to the number of the former rather than the latter. To study the individual roles of the two catalytic sites in a tetramer, we produced mutant PHY-1/PHY-2/(PDI) 2 tetramers in which binding of the Fe 2؉ ion or 2-oxoglutarate to one of the two catalytic sites was prevented. The activities of the mutant tetramers decreased to markedly less than 50% of that of the wild type, being about 5-10% and 20 -30% with the enzymes having one of the two Fe 2؉ -binding sites or 2-oxoglutarate-binding sites inactivated, respectively, while the K m values for these cosubstrates or peptide substrates were not affected. Our data thus indicate that although collagen P4Hs do not act on peptide substrates by a processive mechanism, prevention of hydroxylation at one of the two catalytic sites in the tetramer impairs the function of the other catalytic site.