Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.11.2 (prolyl hydroxylase)
 1,814 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Prolyl 4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha2beta2 tetramers, whereas the Caenorhabditis elegans enzyme is an alphabeta dimer, the beta subunit being identical to protein-disulfide isomerase (PDI). We report here that the processed Drosophila melanogaster alpha subunit is 516 amino acid residues in length and shows 34 and 35% sequence identities to the two types of human alpha subunit and 31% identity to the C. elegans alpha subunit. Its coexpression in insect cells with the Drosophila PDI polypeptide produced an active enzyme tetramer, and small amounts of a hybrid tetramer were also obtained upon coexpression with human PDI. Four of the five recently identified critical residues at the catalytic site were conserved, but a histidine that probably helps the binding of 2-oxoglutarate to the Fe2+ and its decarboxylation was replaced by arginine 490. The enzyme had a higher Km for 2-oxoglutarate, a lower reaction velocity, and a higher percentage of uncoupled decarboxylation than the human enzymes. The mutation R490H reduced the percentage of uncoupled decarboxylation, whereas R490S increased the Km for 2-oxoglutarate, reduced the reaction velocity, and increased the percentage of uncoupled decarboxylation. The recently identified peptide-binding domain showed a relatively low identity to those from other species, and the Km of the Drosophila enzyme for (Pro-Pro-Gly)10 was higher than that of any other animal prolyl 4-hydroxylase studied. A 1. 9-kilobase mRNA coding for this alpha subunit was present in Drosophila larvae.
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PMID:Cloning of the alpha subunit of prolyl 4-hydroxylase from Drosophila and expression and characterization of the corresponding enzyme tetramer with some unique properties. 1003 80

4-Hydroxyproline, the characteristic amino acid of collagens and collagen-like proteins in animals, is also found in certain proline-rich proteins in plants but has been believed to be absent from viral and bacterial proteins. We report here on the cloning and characterization from a eukaryotic algal virus, Paramecium bursaria Chlorella virus-1, of a 242-residue polypeptide, which shows distinct sequence similarity to the C-terminal half of the catalytic alpha subunits of animal prolyl 4-hydroxylases. The recombinant polypeptide, expressed in Escherichia coli, was found to be a soluble monomer and to hydroxylate both (Pro-Pro-Gly)(10) and poly(L-proline), the standard substrates of animal and plant prolyl 4-hydroxylases, respectively. Synthetic peptides such as (Pro-Ala-Pro-Lys)(n), (Ser-Pro-Lys-Pro-Pro)(5), and (Pro-Glu-Pro-Pro-Ala)(5) corresponding to proline-rich repeats coded by the viral genome also served as substrates. (Pro-Ala-Pro-Lys)(10) was a particularly good substrate, with a K(m) of 20 microM. The prolines in both positions in this repeat were hydroxylated, those preceding the alanines being hydroxylated more efficiently. The data strongly suggest that P. bursaria Chlorella virus-1 expresses proteins in which many prolines become hydroxylated to 4-hydroxyproline by a novel viral prolyl 4-hydroxylase.
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PMID:Evidence for 4-hydroxyproline in viral proteins. Characterization of a viral prolyl 4-hydroxylase and its peptide substrates. 1042 73

An efficient expression system for recombinant human collagens will have numerous scientific and medical applications. However, most recombinant systems are unsuitable for this purpose, as they do not have sufficient prolyl 4-hydroxylase activity. We have developed methods for producing the three major fibril-forming human collagens, types I, II and III, in the methylotrophic yeast Pichia pastoris. These methods are based on co-expression of procollagen polypeptide chains with the alpha- and beta-subunits of prolyl 4-hydroxylase. The triple-helical type-I, -II and-III procollagens were found to accumulate predominantly within the endoplasmic reticulum of the yeast cells and could be purified from the cell lysates by a procedure that included a pepsin treatment to convert the procollagens into collagens and to digest most of the non-collagenous proteins. All the purified recombinant collagens were identical in 4-hydroxyproline content with the corresponding non-recombinant human proteins, and all the recombinant collagens formed native-type fibrils. The expression levels using single-copy integrants and a 2 litre bioreactor ranged from 0.2 to 0.6 g/l depending on the collagen type.
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PMID:Expression of recombinant human type I-III collagens in the yeast pichia pastoris. 1096 18

The transmissible spongiform encephalopathies are characterized by conversion of a host protein, PrP(C) (cellular prion protein), to a protease-resistant isoform, PrP(Sc) (prion protein scrapie isoform). The importance of the highly flexible, N-terminal region of PrP has recently become more widely appreciated, particularly the biological activities associated with its metal ion-binding domain and its potential to form a poly(L-proline) II (PPII) helix. Circular dichroism spectroscopy of an N-terminal peptide, PrP(37-53), showed that the PPII helix is formed in aqueous buffer; as it also contains an Xaa-Pro-Gly consensus sequence, it may act as a substrate for the collagen-modifying enzyme prolyl 4-hydroxylase. Direct evidence for this modification was obtained by mass spectrometry and Edman sequencing in recombinant mouse PrP secreted from stably transfected Chinese hamster ovary cells. Almost complete conversion of proline to 4-hydroxyproline occurs specifically at residue Pro44 of this murine protein; the same hydroxylated residue was detected, at lower levels, in PrP(Sc) from the brains of scrapie-infected mice. Cation binding and/or post-translational hydroxylation of this region of PrP may regulate its role in the physiology and pathobiology of the cell.
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PMID:Post-translational hydroxylation at the N-terminus of the prion protein reveals presence of PPII structure in vivo. 1103

Examples of a novel series of phenanthrolinones are shown to be potent competitive inhibitors of avian prolyl 4-hydroxylase, and of collagen hydroxylation, in embryonic chick tendon cells and human foreskin fibroblasts in vitro and in the oestradiol-stimulated rat uterus in vivo. Two compounds, Compound 1 (1,4-dihydrophenanthrolin-4-one-3-carboxylic acid) and Compound 5 [8-(N-butyl-N-ethylcarbamoyl)-1,4-dihydrophenathrolin-4-one-3-carboxylic acid], with comparable potencies in vivo, were chosen to investigate the effect of the inhibition of the hydroxylation of newly synthesized uterine collagen on the turnover of this protein in vivo. Inhibition of hydroxylation by more than 50% for approx. 8 h following single oral doses of the compounds was associated with significant losses of radiolabelled proline and 4-hydroxyproline from collagen during this period. Progressive hydroxylation of collagen over 48 h, as the inhibitory action of the compounds declined, was accompanied by a decreased loss of radiolabel from the uterine collagen. Earlier reports indicated that underhydroxylated collagen, accumulating within the endoplasmic reticulum in cells where prolyl 4-hydroxylase is inactivated, is slowly degraded, but is then rapidly hydroxylated and secreted when the activity of prolyl 4-hydroxylase is restored. Taken with the present results, this suggests that the potential use of inhibitors of prolyl 4-hydroxylase to control excessive collagen deposition in pathological fibrosis may be limited by the need to maintain continuous inhibition of collagen hydroxylation so as to facilitate intracellular degradation of the accumulated protein.
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PMID:Inhibition of prolyl 4-hydroxylase in vitro and in vivo by members of a novel series of phenanthrolinones. 1113 98

The extracellular matrix (ECM) is proposed to play critical roles in organ morphogenesis through the stabilization and/or sequestration of signaling factors and adhesion molecules, and by maintaining organ integrity. As a first step toward understanding molecules involved in ECM modification and maturation, we have examined the embryonic expression profiles of ten prolyl 4-hydroxylase alpha subunit (PH4alpha)-related genes. Prolyl 4-hydroxylases (PH4) catalyze the formation of 4-hydroxyproline in collagens, the major components of the ECM, and are implicated in the hydroxylation of proline in several other secreted proteins. To date, two alpha subunit proteins have been described in both humans (PHalphaI and PHalphaII) and worms (PHY-1/DPY-18 and PHY-2), whereas only a single Drosophila alpha subunit has been identified. The ten PH4alpha-related genes described in this study are clustered in a 183-kb region near the tip of chromosome arm 3R and include the previously described Drosophila alpha subunit gene. Six of the ten PH4alpha genes in the cluster have tissue-specific embryonic expression. PH4alphaSG1 and PH4alphaSG2 are expressed in the salivary gland, PH4alphaMP is expressed in mouth-part precursors, PH4alphaPV is expressed in the proventriculus, and CG9698-E is expressed in the epidermis. PH4alphaEFB is expressed more broadly, with expression in the anterior and posterior midgut primordia, the fat body, the hemocytes and the epidermis. The expression profiles of these PH4alpha-related genes suggest that tissue-specific ECM modifications may be critical to organ formation and/or function.
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PMID:Prolyl 4-hydroxylase alpha-related proteins in Drosophila melanogaster: tissue-specific embryonic expression of the 99F8-9 cluster. 1185 Jan 89

The collagen prolyl 4-hydroxylases (EC ) play a critical role in the synthesis of all collagens. The enzymes from all vertebrate species studied are alpha(2)beta(2) tetramers, in which the beta subunit is identical to protein disulfide isomerase (PDI). Two isoforms of the catalytic alpha subunit, PHY-1 and PHY-2, have previously been characterized from Caenorhabditis elegans. We report here on the cloning and characterization of a third C. elegans alpha subunit isoform, PHY-3. It is much shorter than the previously characterized vertebrate and C. elegans alpha subunits and shows 23-30% amino acid sequence identity to PHY-1 and PHY-2 within the catalytic C-terminal region. Recombinant PHY-3 coexpressed in insect cells with a C. elegans PDI isoform that does not associate with PHY-1 was found to be an active prolyl 4-hydroxylase. The phy-3 gene consists of five exons, and its expression pattern differs distinctly from the hypodermally expressed phy-1 and phy-2 in that it is expressed in embryos, late larval stages, and adult nematodes, expression in the latter being restricted to the spermatheca. Nematodes homozygous for a phy-3 deletion are phenotypically of the wild type and fertile, but the 4-hydroxyproline content of phy-3(-/-) early embryos was reduced by about 90%. PHY-3 is thus likely to be involved in the synthesis of collagens in early embryos, probably of those in the egg shell.
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PMID:Egg shell collagen formation in Caenorhabditis elegans involves a novel prolyl 4-hydroxylase expressed in spermatheca and embryos and possessing many unique properties. 1189 Dec 26

At least 28 proteins have now been defined as collagens (Trends Genet. 20:33-43, 2004; J. Biol. Chem. 281:3494-3504, 2006), but many of those recently discovered are present in tissues in such small amounts that their isolation for characterization at the protein level has so far been impossible. Some of the fibrilforming collagens are used as a biomaterial in numerous medical applications and as a delivery system for various drugs (3, 4). The collagens used in all these applications have been isolated from animal tissues and are liable to cause allergic reactions in some subjects and carry a risk of disease-causing contaminants (3,4). An efficient recombinant expression system for collagens can thus be expected to have numerous scientific and medical applications. The systems commonly used for expressing other proteins in lower organisms are not suitable as such for the production of recombinant collagens, however, as bacteria and yeast have no prolyl 4-hydroxylase activity and insect cells have insufficient levels of it. Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer in vertebrates, plays a central role in the synthesis of all collagens, as 4-hydroxyproline-deficient collagen polypeptide chains cannot form triple helices that are stable at 37 degrees C (5,6). All attempts to assemble an active prolyl 4-hydroxylase tetramer from its subunits in vitro have been unsuccessful, but active recombinant human prolyl 4-hydroxylase has been produced in insect cells, yeast, and Escherichia coli by coexpression of its alpha - and beta -subunits (7-9).
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PMID:Recombinant collagen trimers from insect cells and yeast. 1924 4

Posttranslational modifications can cause profound changes in protein function. Typically, these modifications are reversible, and thus provide a biochemical on-off switch. In contrast, proline residues are the substrates for an irreversible reaction that is the most common posttranslational modification in humans. This reaction, which is catalyzed by prolyl 4-hydroxylase (P4H), yields (2S,4R)-4-hydroxyproline (Hyp). The protein substrates for P4Hs are diverse. Likewise, the biological consequences of prolyl hydroxylation vary widely, and include altering protein conformation and protein-protein interactions, and enabling further modification. The best known role for Hyp is in stabilizing the collagen triple helix. Hyp is also found in proteins with collagen-like domains, as well as elastin, conotoxins, and argonaute 2. A prolyl hydroxylase domain protein acts on the hypoxia inducible factor alpha, which plays a key role in sensing molecular oxygen, and could act on inhibitory kappaB kinase and RNA polymerase II. P4Hs are not unique to animals, being found in plants and microbes as well. Here, we review the enzymic catalysts of prolyl hydroxylation, along with the chemical and biochemical consequences of this subtle but abundant posttranslational modification.
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PMID:Prolyl 4-hydroxylase. 2019 58

Prolyl 4-hydroxylases are ascorbate-dependent oxygenases that play key roles in a variety of eukaryotic biological processes including oxygen sensing, siRNA regulation, and collagen folding. They perform their functions by catalyzing the post-translational hydroxylation of specific proline residues on target proteins to form (2S,4R)-4-hydroxyproline. Thus far, the study of these post-translational modifications has been limited by the lack of a prokaryotic recombinant expression system for producing hydroxylated proteins. By introducing a biosynthetic shunt to produce ascorbate-like molecules in Eschericia coli cells that heterologously express human prolyl 4-hydroxylase (P4H), we have created a strain of E. coli that produces collagenous proteins with high levels of (2S,4R)-4-hydroxyproline. Using this new system, we have observed hydroxylation patterns indicative of a processive catalytic mode for P4H that is active even in the absence of ascorbate. Our results provide insights into P4H enzymology and create a foundation for better understanding how post-translational hydroxylation affects proteins.
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PMID:Tunable, post-translational hydroxylation of collagen Domains in Escherichia coli. 2121 Jun 82


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