EC:1.14.11.2 - FACTA Search

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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 (EC 1.14.11.2) catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer, the beta subunit of which is a highly unusual multifunctional polypeptide, being identical to protein disulfide-isomerase (EC 5.3.4.1). We report here the cloning of a second mouse alpha subunit isoform, termed the alpha (II) subunit. This polypeptide consists of 518 aa and a signal peptide of 19 aa. The processed polypeptide is one residue longer than the mouse alpha (I) subunit (the previously known type), the cloning of which is also reported here. The overall amino acid sequence identity between the mouse alpha (II) and alpha (I) subunits is 63%. The mRNA for the alpha (II) subunit was found to be expressed in a variety of mouse tissues. When the alpha (II) subunit was expressed together with the human protein disulfide-isomerase/beta subunit in insect cells by baculovirus vectors, an active prolyl 4-hydroxylase was formed, and this protein appeared to be an alpha (II) 2 beta 2 tetramer. The activity of this enzyme was very similar to that of the human alpha (I) 2 beta 2 tetramer, and most of its catalytic properties were also highly similar, but it differed distinctly from the latter in that it was inhibited by poly(L-proline) only at very high concentrations. This property may explain why the type II enzyme was not recognized earlier, as an early step in the standard purification procedure for prolyl 4-hydroxylase is affinity chromatography on a poly(L-proline) column.
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PMID:Cloning, baculovirus expression, and characterization of a second mouse prolyl 4-hydroxylase alpha-subunit isoform: formation of an alpha 2 beta 2 tetramer with the protein disulfide-isomerase/beta subunit. 775 22

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer, the beta subunit of which is identical to protein disulfide-isomerase (PDI). We report here on the cloning of the catalytically important alpha subunit from Caenorhabditis elegans. This polypeptide consists of 542 amino acids and signal peptide of 16 additional residues. The C. elegans alpha subunit is 25 amino acids longer than the human alpha subunit, mainly because of a 32-amino-acid C-terminal extension present only in the former. The overall amino acid sequence identity between these two alpha subunits is 45%, a 127-amino acid region close to the C terminus being especially well conserved. When the C. elegans alpha subunit was expressed together with the human PDI/beta subunit in insect cells by baculovirus vectors, an active prolyl 4-hydroxylase was formed, but surprisingly this C. elegans/human enzyme appeared to be an alpha beta dimer. The specific activity of this C. elegans/human enzyme was comparable with that of the human enzyme, and most of the other catalytic properties were also highly similar. Nevertheless, the C. elegans/human enzyme was not inhibited by poly(L-proline). The data indicate that the multifunctional PDI/beta subunit can form an active prolyl 4-hydroxylase with alpha subunits having marked differences in their amino acid sequences.
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PMID:Cloning, baculovirus expression, and characterization of the alpha subunit of prolyl 4-hydroxylase from the nematode Caenorhabditis elegans. This alpha subunit forms an active alpha beta dimer with the human protein disulfide isomerase/beta subunit. 792 9

The protein or cDNA sequencing revealed that the membrane-associated 3,5,3'-triiodo-thyronine binding protein (T3BP) acts as a multifunctional protein:protein disulfide isomerase (PDI) catalyzing isomerization of intra- and inter-molecular disulfide bridge in the proteins, beta-subunit of prolyl 4-hydroxylase catalyzing the formation of 4-hydroxyproline in collagen molecules, glycosylation site binding protein which is a component of oligosaccharyl transferase transferring oligosaccharide chains to the asparagine residues of Asn-X-Ser/Thr site in nascent polypeptide, and a component of triglyceride transfer protein complex involved in the transfer unit of triglyceride, cholesteryl ester and phosphatidylcholine between biomembranes. The functions of 55 k-T3BP/PDI, mainly involved in important post-translational modifications, are discussed in relation to the domain structure of the molecule.
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PMID:[Molecular cloning and multifunctions of membrane-associated 3,5,3'-triiodo-L-thyronine binding protein with protein disulfide isomerase activity]. 819 76

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyses the post-translational formation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha2beta2 tetramers, their beta subunit being identical to protein disulphide isomerase (PDI). The function of the PDI-beta subunit in prolyl 4-hydroxylases is not fully understood, but it seems to be that of keeping the highly insoluble alpha subunits in solution. We report here that expression of the alpha subunit of human type I prolyl 4-hydroxylase in insect cells together with BiP polypeptide leads to the formation of both soluble and insoluble alpha-subunit-BiP complexes. Formation of the soluble complexes was evident from (1) a marked increase in the amount of the alpha subunit in the soluble fraction of the cell homogenates when expressed together with BiP, (2) immunoprecipitation experiments and (3) demonstration of the presence of some of the complexes by polyacrylamide gel electrophoresis under non-denaturing conditions. Formation of the insoluble complexes was suggested by an increase in the amount of BiP in the insoluble fraction when expressed together with the alpha subunit. Nevertheless the soluble alpha-subunit-BiP complexes had no prolyl 4-hydroxylase activity. This indicates that the function of the PDI-beta subunit in the prolyl 4-hydroxylase tetramer is not only that of keeping the alpha subunits in solution but appears to be more specific, probably that of keeping them in a catalytically active, non-aggregated conformation.
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PMID:Co-expression of the alpha subunit of human prolyl 4-hydroxylase with BiP polypeptide in insect cells leads to the formation of soluble and insoluble complexes. Soluble alpha-subunit-BiP complexes have no prolyl 4-hydroxylase activity. 861 37

An efficient expression system for recombinant collagens would have numerous scientific and practical applications. Nevertheless, most recombinant systems are not suitable for this purpose, as they do not have sufficient amounts of prolyl 4-hydroxylase activity. Pro-alpha 1 chains of human type III collagen expressed in insect cells by a baculovirus vector are reported here to contain significant amounts of 4-hydroxyproline and to form triple-helical molecules, although the Tm of the triple helices was only about 32-34 degrees C. Coexpression of the pro-alpha1(III) chains with the alpha and beta subunits of human prolyl 4-hydroxylase increased the Tm to about 40 degrees C, provided that ascorbate was added to the culture medium. The level of expression of type III procollagen was also increased in the presence of the recombinant prolyl 4-hydroxylase, and the pro-alpha 1(III) chains and alpha1(III) chains were found to be present in disulfide-bonded molecules. Most of the triple-helical collagen produced was retained within the insect cells and could be extracted from the cell pellet. The highest expression levels were obtained in High Five cells, which produced up to about 80 microg of cellular type III collagen (120 microg of procollagen) per 5 X 10(6) cells in monolayer culture and up to 40 mg/liter of cellular type III collagen (60 mg/liter procollagen) in suspension. The 4-hydroxyproline content and Tm of the purified recombinant type III collagen were very similar to those of the nonrecombinant protein, but the hydroxylysine content was slightly lower, being about 3 residues/1000 in the former and 5/1000 in the latter.
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PMID:Characterization of human type III collagen expressed in a baculovirus system. Production of a protein with a stable triple helix requires coexpression with the two types of recombinant prolyl 4-hydroxylase subunit. 866 31

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyses the formation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha 2 beta 2 tetramers while the Caenorhabditis elegans enzyme is an alpha beta dimer. The beta-subunit is identical to protein disulphide isomerase (PDI), a multifunctional endoplasmic reticulum luminal polypeptide. ERp60 is a PDI isoform that was initially misidentified as a phosphatidylinositol-specific phospholipase C. We report here on the cloning and expression of the human and Drosophila ERp60 polypeptides. The overall amino acid sequence identity and similarity between the processed human ERp60 and PDI polypeptides are 29% and 56% respectively, and those between the Drosophila ERp60 and human PDI polypeptides 29% and 55%. The two ERp60 polypeptides were found to be similar to human PDI within almost all their domains, the only exception being the extreme C-terminal region. Nevertheless, when the human or Drosophila ERp60 was expressed in insect cells together with an alpha-subunit of human prolyl 4-hydroxylase, no tetramer was formed and no prolyl 4-hydroxylase activity was generated in the cells. Additional experiments with hybrid polypeptides in which the C-terminal regions had been exchanged between the human ERp60 and PDI polypeptides demonstrated that the differences in the C-terminal region are not the only reason for the lack of prolyl 4-hydroxylase tetramer formation by ERp60.
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PMID:ERp60 does not substitute for protein disulphide isomerase as the beta-subunit of prolyl 4-hydroxylase. 868 6

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha2beta2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens. We converted 16 residues in the human alpha subunit individually to other amino acids, and expressed the mutant polypeptides together with the wild-type beta subunit in insect cells. Asp414Ala and Asp414Asn inactivated the enzyme completely, whereas Asp414Glu increased the K(m) for Fe2+ 15-fold and that for 2-oxoglutarate 5-fold. His412Glu, His483Glu and His483Arg inactivated the tetramer completely, as did Lys493Ala and Lys493His, whereas Lys493Arg increased the K(m) for 2-oxoglutarate 15-fold. His501Arg, His501Lys, His501Asn and His501Gln reduced the enzyme activity by 85-95%; all these mutations increased the K(m) for 2-oxoglutarate 2- to 3-fold and enhanced the rate of uncoupled decarboxylation of 2-oxoglutarate as a percentage of the rate of the complete reaction up to 12-fold. These and other data indicate that His412, Asp414 and His483 provide the three ligands required for the binding of Fe2+ to a catalytic site, while Lys493 provides the residue required for binding of the C-5 carboxyl group of 2-oxoglutarate. His501 is an additional critical residue at the catalytic site, probably being involved in both the binding of the C-1 carboxyl group of 2-oxoglutarate and the decarboxylation of this cosubstrate.
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PMID:Characterization of the iron- and 2-oxoglutarate-binding sites of human prolyl 4-hydroxylase. 913 34

Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha2beta2 tetramer, the beta subunit of which is identical to protein disulfide-isomerase (PDI, EC 5.3.4.1). We report here on cloning of the recently discovered alpha(II) subunit from human sources. The mRNA for the alpha(II) subunit was found to be expressed in a variety of human tissues, and the presence of the corresponding polypeptide and the (alpha(II))2beta2 tetramer was demonstrated in cultured human WI-38 and HT-1080 cells. The type II tetramer was found to represent about 30% of the total prolyl 4-hydroxylase in these cells and about 5-15% in various chick embryo tissues. The results of coexpression in insect cells argued strongly against the formation of a mixed alpha(I)alpha(II)beta2 tetramer. PDI/beta polypeptide containing a histidine tag in its N terminus was found to form prolyl 4-hydroxylase tetramers as readily as the wild-type PDI/beta polypeptide, and histidine-tagged forms of prolyl 4-hydroxylase appear to offer an excellent source for a simple large scale purification of the recombinant enzyme. The properties of the purified human type II enzyme were very similar to those of the type I enzyme, but the Ki of the former for poly(L-proline) was about 200-1000 times that of the latter. In agreement with this, a minor difference, about 3-6-fold, was found between the two enzymes in the Km values for three peptide substrates. The existence of two forms of prolyl 4-hydroxylase in human cells raises the possibility that mutations in one enzyme form may not be lethal despite the central role of this enzyme in the synthesis of all collagens.
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PMID:Cloning of the human prolyl 4-hydroxylase alpha subunit isoform alpha(II) and characterization of the type II enzyme tetramer. The alpha(I) and alpha(II) subunits do not form a mixed alpha(I)alpha(II)beta2 tetramer. 921 72

Prolyl 4-hydroxylase, the key enzyme of collagen synthesis, is an alpha2beta2 tetramer, the beta subunit of which is protein disulfide isomerase (PDI). Coexpression of the human alpha subunit and PDI in Pichia produced trace amounts of an active tetramer. A much higher, although still low, assembly level was obtained using a Saccharomyces pre-pro sequence in PDI. Coexpression with human type III procollagen unexpectedly increased the assembly level 10-fold, with no increase in the total amounts of the subunits. The recombinant enzyme was active not only in Pichia extracts but also inside the yeast cell, indicating that Pichia must have a system for transporting all the cosubstrates needed by the enzyme into the lumen of the endoplasmic reticulum. The 4-hydroxyproline-containing procollagen polypeptide chains were of full length and formed molecules with stable triple helices even though Pichia probably has no Hsp47-like protein. The data indicate that collagen synthesis in Pichia, and probably also in other cells, involves a highly unusual control mechanism, in that production of a stable prolyl 4-hydroxylase requires collagen expression while assembly of a stable collagen requires enzyme expression. This Pichia system seems ideal for the high-level production of various recombinant collagens for numerous scientific and medical purposes.
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PMID:Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase. 936 85

Prolyl 4-hydroxylases (EC 1.14,11.2) catalyze the formation of 4-hydroxyproline in collagens and other proteins with collagen-like sequences. The vertebrate type I and type II enzymes are [alpha (I)]2 beta 2 and [alpha (II)]2 beta 2 tetramers, respectively, whereas the enzyme from the nematode Caenorhabditis elegans is an alpha beta dimer. The type I enzyme is the major form in most but not all vertebrate tissues. The catalytic properties of the various enzyme forms are highly similar, but there are distinct, although small, differences in K(m) values for various peptide substrates between the enzyme forms and major differences in Ki values for the competitive inhibitor, poly(L-proline). Prolyl 4-hydroxylase requires Fe2+, 2-oxoglutarate, O2 and ascorbate. Kinetic studies and theoretical considerations have led to elucidation of the reaction mechanism, and recent extensive site-directed mutagenesis studies have identified five critical residues at the cosubstrate binding sites. A number of compounds have been characterized that inhibit it competitively with respect to some of the cosubstrates, and three groups of suicide inactivators have also been identified. The beta subunit in all forms of prolyl 4-hydroxylase is identical to protein disulfide isomerase (PDI), a multifunctional polypeptide that also serves as a subunit in the microsomal triglyceride transfer protein, as a chaperone-like polypeptide that probably assists folding of a number of newly synthesized proteins, and in several other functions. The main role of the PDI polypeptide as a protein subunit is probably related to its chaperone function. Recent expression studies of recombinant human prolyl 4-hydroxylase subunits in a yeast have indicated that the formation of a stable enzyme tetramer in vivo requires coexpression of collagen polypeptide chains.
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PMID:Prolyl 4-hydroxylases and their protein disulfide isomerase subunit. 952 56

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