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J. Biol. Chem., Vol. 277, Issue 26, 23965-23971, June 28, 2002

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Cloning and Characterization of a Low Molecular Weight Prolyl 4-Hydroxylase from Arabidopsis thaliana

EFFECTIVE HYDROXYLATION OF PROLINE-RICH, COLLAGEN-LIKE, AND HYPOXIA-INDUCIBLE TRANSCRIPTION FACTOR -LIKE PEPTIDES^*

Reija Hieta and Johanna Myllyharju

From the Collagen Research Unit, Biocenter Oulu and the Department of Medical Biochemistry and Molecular Biology, University of Oulu, FIN-90014 Oulu, Finland

Received for publication, February 25, 2002, and in revised form, April 25, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

4-Hydroxyproline is found in collagens and collagen-like proteins in animals and in many glycoproteins in plants. Animal prolyl 4-hydroxylases (P4Hs) have been cloned and characterized from many sources, but no plant P4H has been cloned so far. We report here that the genome of Arabidopsis thaliana encodes six P4H-like polypeptides, one of which, a 283-residue soluble monomer, was cloned and characterized here as a recombinant protein. Catalytically critical residues identified in animal P4Hs are conserved in this P4H, and their mutagenesis led to complete or almost complete inactivation. The recombinant P4H effectively hydroxylated poly(L-proline) and many synthetic peptides corresponding to proline-rich repeats present in plant glycoproteins and other proteins. Surprisingly, collagen-like peptides were also good substrates, the V_max with (Pro-Pro-Gly)₁₀ being similar to that with poly(L-proline). The enzyme acted in this peptide preferentially on prolines in Y positions in the X-Y-Gly triplets. Correspondingly, (Gly-Pro-4Hyp)₅ and (Pro-Ala-Gly)₅ were poor substrates, with V_max values less than 5 and 20% of that obtained with (Pro-Pro-Gly)₁₀, respectively, the K_m for the latter also being high. Peptides representing the N- and C-terminal hydroxylation sites present in hypoxia-inducible transcription factor  also served as substrates. As these peptides contain only one proline residue, a poly(L-proline) type II conformation was clearly not required for hydroxylation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

4-Hydroxyproline is found in collagens, elastin, and more than 15 additional proteins with collagen-like domains in animal tissues (1-3). Its formation is catalyzed by prolyl 4-hydroxylases (P4Hs),¹ which act within the lumen of the endoplasmic reticulum and hydroxylate -X-Pro-Gly- sequences. The reaction requires Fe²⁺, 2-oxoglutarate, O₂, and ascorbate and involves an oxidative decarboxylation of 2-oxoglutarate (for reviews, see Refs. 3 and 4). The vertebrate enzymes are ₂₂ tetramers with a molecular weight of approximately 240,000, in which the  subunit is identical to the enzyme and chaperone protein-disulfide isomerase (3, 4). Two isoforms of the catalytic  subunit have been cloned and extensively characterized from several sources and shown to form [(I)₂]₂ and [(II)₂]₂ tetramers with protein-disulfide isomerase (3-6). Animal P4Hs have also been cloned and characterized from Caenorhabditis elegans (7-11) and Drosophila melanogaster (12). In addition, a family of cytoplasmic P4Hs that hydroxylate proline in -Leu-X-X-Leu-Ala-Pro- sequences has very recently been found to play a critical role in the regulation of the hypoxia-inducible transcription factor (HIF)  (13-16).

4-Hydroxyproline is also found in many plant glycoproteins, especially in extensins, proline-rich glycoproteins, and arabinogalactan proteins (17-20). P4Hs from unicellular and multicellular green algae are 60-kDa monomers (21, 22). Those from higher plants are also likely to be monomers, although the variable presence of an additional polypeptide has been reported in partially purified preparations (23, 24). No plant P4H has been cloned and characterized in detail so far, however. Plant P4Hs require the same cosubstrates as the animal enzymes, but they differ from them in that they act primarily on poly(L-proline)-like sequences and may require the poly(L-proline) II helix (for a review, see Ref. 25). Very low hydroxylation rates have also been reported with random-coil but not triple-helical forms of (Pro-Pro-Gly)₅ and (Pro-Pro-Gly)₁₀, however (25). Recently, a viral P4H has been cloned and characterized from Paramecium bursaria chlorella virus-1 (PBCV-1) (26). This enzyme is a 242-amino acid monomer that resembles plant P4Hs in that it hydroxylates poly(L-proline) and (Pro-Pro-Gly)₁₀, the latter with a much higher K_m (26). The recombinant viral P4H also hydroxylates many synthetic peptides corresponding to proline-rich repeats coded by the viral genome (26).

Our sequence homology searches of the Arabidopsis thaliana genome indicated that it contains six open reading frames coding for P4H-like polypeptides. We have now cloned one of these, which encodes a 283-amino acid polypeptide. The recombinant enzyme expressed in insect cells was found to be a monomer that hydroxylated poly(L-proline) and many other proline-rich peptides. Surprisingly, it also effectively hydroxylated the collagen-like peptides (Pro-Pro-Gly)₁₀ and (Ala-Pro-Gly)₅ with K_m values that are similar to those reported for animal P4Hs. Furthermore, the recombinant A. thaliana P4H resembled the animal enzymes in that it preferentially hydroxylated proline residues preceding glycines in (X-Y-Gly)_n peptides.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Identification of A. thaliana Genes Encoding P4H-like Polypeptides-- A sequence homology search (27) of the A. thaliana genome indicated the presence of six genes encoding polypeptides of 280-332 amino acids (GenBank^TM accession nos. AAC64297, AAB80790, AAF88161, NP_197391, AAF08583, and BAB02864) with similarity to the catalytic C-terminal halves of the human P4H (I) and (II) subunits (6, 28). These amino acid sequences were aligned (29) with those of the human (I) and (II) subunits and the PBCV-1 P4H (26), and the cleavage sites of the signal peptides were predicted (30).

Cloning and Expression of a Recombinant A. thaliana P4H in Insect Cells-- PCR primers 5'-GCGGGATCCCTCCTTGTTACAATTGGCCTTTA-3' and 5'-CGGGATCCTCAAGAAGTAGCTTTTTGCCTCAT-3' were synthesized based on the gene encoding the polypeptide AAC64297 (named here At-P4H-1) and used to obtain a 783-base pair PCR product from a whole plant A. thaliana cDNA library (Stratagene). The PCR template was prepared by incubating 2 µl of the cDNA library in a 200-µl final volume in 1% Nonidet P-40, 100 µg/ml proteinase K, 1 mM EDTA, 10 mM Tris, pH 8.0, at 55 °C for 45 min, followed by 10 min at 95 °C, centrifuged at 12,000 rpm for 5 min, and 10 µl of the prepared template was used in a 50-µl PCR reaction. Hot-start PCR with preincubation for 5 min at 94 °C and 2 min at 72 °C before the addition of 2 µl of Pfu polymerase (Promega) was used, after which 30 PCR cycles were performed as follows: denaturation for 1 min at 94 °C, annealing for 2 min at 65 °C, and extension for 3 min at 72 °C. To increase the amount of the obtained PCR product, a second PCR reaction was performed with 10 µl of 1:50 diluted first PCR reaction product as the template. The PCR cycles were as above, with the exception that the annealing temperature was 58 °C. The obtained PCR fragment coding for residues Ser²³-Ser²⁸³ of At-P4H-1 had BamHI restriction sites at both ends (underlined in the primer sequences) and one cytosine before the codon for Ser²³, and it was cloned into a BamHI-digested baculovirus vector pACGP67-A (Invitrogen). The sequences were verified on an automated DNA sequencer (Abi Prism 377, Applied Biosystems).

The recombinant vector was cotransfected into Spodoptera frugiperda Sf9 cells with BaculoGold DNA (PharMingen) by calcium phosphate transfection, and the recombinant viruses were amplified (31). Sf9 or High Five insect cells (Invitrogen) were cultured as monolayers in TNM-FH medium (Sigma) supplemented with 10% fetal bovine serum (BioClear) or in suspension in Sf900IISFM serum-free medium (Invitrogen). The cells were seeded at a density of 5 × 10⁶ cells/100-mm plate or 1 × 10⁶/ml and infected at a multiplicity of 5 with the virus coding for the At-P4H-1. The cells were harvested 72 h after infection, washed with a solution of 0.15 M NaCl and 0.02 M phosphate, pH 7.4, and homogenized in a 0.1 M NaCl, 0.1 M glycine, 10 µM dithiothreitol (DTT), 0.1% or 1% Triton X-100, and 0.01 M Tris buffer, pH 7.8, or in a 50% glycerol, 0.6 M NaCl, 1% Nonidet P-40, 0.1 M glycine, 100 µM DTT, and 0.06 M Tris buffer, pH 7.8, and centrifuged at 10,000 × g for 20 min. The pellets were further solubilized in 1% SDS, and aliquots of all soluble fractions were analyzed by 12% SDS-PAGE under reducing conditions.

Expression of a Recombinant A. thaliana P4H in Escherichia coli-- PCR primers 5'-GGAATTCCATATGTCCTTGTTACAATTGGCCTTTAT-3' and 5'-CGGGATCCTCAAGAAGTAGCTTTTTGCCTCAT-3' were used to amplify the At-P4H-1 cDNA without the signal sequence and with flanking NdeI and BamHI sites as above, and the product was cloned into a NdeI-BamHI-digested expression vector pET15b (Novagen).

The expression plasmid was transformed into the E. coli BL21(DE3) strain (Novagen). The cells were grown at 37 °C to an optical density of 0.5 at 600 nm, incubated at 30 °C for 30 min, and expression was induced with 1 mM isopropyl-1-thio--D-galactopyranoside. The cells were harvested 3 h after induction, suspended in 0.1 volume of 50 mM Tris-HCl, pH 8.0, with or without 0.1% Triton X-100, sonicated, centrifuged at 17,000 × g for 20 min, and the soluble and insoluble fractions analyzed by 12% SDS-PAGE.

Site-directed Mutagenesis-- Histidines 180 and 260 in the At-P4H-1 were converted individually to glutamate (codon GAA) and alanine (GCT), Asp¹⁸² to alanine (GCT) and glutamate (GAA), Lys²⁷⁰ to arginine (AGG) and alanine (GCG), Ser²⁷² to alanine (GCT), and Arg²⁷⁸ to alanine (GCG) and histidine (CAC). The mutagenesis reactions were performed in the pET15b vector containing the full-length At-P4H-1 cDNA using a QuikChange^TM site-directed mutagenesis kit (Stratagene). The mutant cDNAs were amplified by PCR using the primers with flanking BamHI sites (above), and the products were digested with BamHI and cloned into BamHI-digested pACGP67-A. Recombinant baculoviruses were generated and used to infect insect cells as above.

Other Assays-- P4H activity was assayed at 30 °C by a method based on the hydroxylation-coupled decarboxylation of 2-oxo-[1-¹⁴C]glutarate (32). Poly(L-proline) was purchased from Sigma, whereas all the other synthetic peptides were from Innovagen. All the peptides except poly(L-proline) and those representing HIF sequences were denatured by heating to 100 °C for 10 min, followed by rapid cooling before addition to the enzyme reaction mixture. In some experiments the amount of 4-hydroxyproline formed was determined by a colorimetric method in samples hydrolyzed with 6 M HCl at 120 °C overnight (33), and in others the partially hydroxylated (Pro-Pro-Gly)₁₀ peptide was purified from the reaction mixture with HiTrap Q-Sepharose (Amersham Biosciences) and reverse phase HPLC, hydrolyzed by the manual gas-phase hydrolysis method, and analyzed in an Applied Biosystems 421A amino acid analyzer. N-terminal sequencing of the purified (Pro-Pro-Gly)₁₀ peptide was performed in an Applied Biosystems 492 Procise^TM protein sequencer. Typically, approximately one third of the amount of proline and 4-hydroxyproline present in each sequencing cycle was carried over to the next cycle. The values obtained were corrected for this carry over, but they were not corrected for a significant background level, as this could not be quantitated accurately. K_m values were determined as described previously (34). The molecular weight of the recombinant At-P4H-1 was analyzed by gel filtration in a calibrated HiPrep Sephacryl S-100 HR column (Amersham Biosciences).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The A. thaliana Genome Encodes Several P4H-like Polypeptides-- A sequence homology search indicated that the A. thaliana genome contains six open reading frames encoding polypeptides of 280-332 residues (Fig. 1) that show an identity of 21-27% to the catalytically important C-terminal regions of the human P4H (I) and (II) subunits (6, 28). The six polypeptides are 33-81% identical to each other (Fig. 1). The two histidines and one aspartate that bind the Fe²⁺ atom at the catalytic site (34, 35) and the lysine that binds the C-5 carboxyl group of the 2-oxoglutarate in all collagen P4Hs (34) are all conserved in the A. thaliana sequences (Fig. 1). The fifth critical residue, a histidine that is probably involved in the binding of the C-1 carboxyl group of 2-oxoglutarate to the Fe²⁺ atom and the decarboxylation of this cosubstrate (34), is conserved in five of the sequences, but is replaced by an arginine in the AAC64297 polypeptide. This position is also occupied by an arginine in a Drosophila P4H  subunit (12) and in the PBCV-1 viral P4H (26), suggesting that all six A. thaliana polypeptides are P4Hs. However, like the PBCV-1 enzyme, these polypeptides show no similarity to the peptide substrate binding domain that is located between residues 140 and 240 in the animal P4H  subunits (36). A noncleavable signal peptide was predicted in the AAC64297 polypeptide and cleavable ones in the AAF88161, NP_197391 and AAF08583 polypeptides, whereas no signal peptide was present in AAB80790 and BAB02864 (Fig. 1). The sequence identity of the 283-amino acid polypeptide AAC64297 to the human (I) and (II) subunits was highest, 25 and 27%, respectively. The cDNA encoding this polypeptide, named At-P4H-1, was cloned and recombinantly expressed. At-P4H-1 has four cysteine residues, the first being conserved in all six A. thaliana P4H-like polypeptides and the fourth, in position +3 with respect to the second Fe²⁺-binding histidine, also being conserved in the human P4H  subunits. Two of the cysteines are not conserved in the six A. thaliana polypeptides and are present in a region that has no homologous counterpart in the PBCV-1 P4H or the human  subunits (Fig. 1). At-P4H-1 has no potential N-glycosylation sites, whereas the other A. thaliana P4H-like polypeptides have one to four such sites.

View larger version (69K): [in this window] [in a new window]   Fig. 1.   Alignment of the amino acid residues in the six A. thaliana P4H-like polypeptides, PBCV-1 viral P4H, and the (I) subunit of human type I P4H. The residues present in the human (I) subunit that precede the alignment region are not shown. The six A. thaliana P4H-like polypeptides are indicated by their GenBankTM accession nos. AAC64297 (named At-P4H-1), AAB80790, AAF88161, NP_197391, AAF08583, and BAB02864, the P. bursaria chlorella virus-1 P4H by PBCV-1, and the human (I) subunit by H(I). Amino acids that are identical between two of the polypeptides are shown with black backgrounds. Gaps were introduced for maximal alignment. The three Fe2+-binding residues, two histidines and one aspartate, the lysine that binds the C-5 carboxyl group of 2-oxoglutarate, and the serine and arginine in positions +2 and +8 from the lysine, respectively, are indicated by asterisks.

Expression of a Recombinant A. thaliana P4H-like Polypeptide in Insect Cells and E. coli-- A cDNA encoding At-P4H-1 residues Ser²³-Ser²⁸³ was synthesized by PCR, cloned into the baculovirus vector pACGP67-A in frame with the GP67 signal sequence, and used to generate a recombinant virus. The cells infected with this virus were harvested 72 h after infection, homogenized in a buffer containing 0.1% Triton X-100, and centrifuged. The remaining pellet was further solubilized in 1% SDS, and the samples were analyzed by SDS-PAGE and Coomassie Blue staining (Fig. 2). Very little of the recombinant 29-kDa polypeptide was extracted with 0.1% Triton X-100 (Fig. 2, lane 1), whereas most of it remained in the insoluble fraction and could be extracted with 1% SDS (Fig. 2, lane 4). Therefore, various means of extracting the polypeptide more efficiently were tested, including sonication, the use of buffers with high salt concentrations, various detergents, and low concentrations of urea (details not shown). A buffer containing 1% Triton X-100 slightly improved the solubility (Fig. 2, lane 2), whereas a solution consisting of 50% glycerol, 0.6 M NaCl, 1% Nonidet P-40, 0.1 M glycine, 100 µM DTT, and 0.06 M Tris, pH 7.8 (37), was found to be the best solubilizing method among those tested (Fig. 2, lane 3), although even this solubilized only approximately 10% of the enzyme.

View larger version (80K): [in this window] [in a new window]   Fig. 2.   Analysis of the expression of the At-P4H-1 polypeptide in insect cells by SDS-PAGE under reducing conditions. A recombinant pAcGP67 vector coding for amino acids Ser23-Ser283 of the At-P4H-1 was used to infect insect cells. The cells were harvested 72 h after infection, homogenized in a buffer containing 0.1% (lane 1) or 1% (lane 2) Triton X-100, or 50% glycerol, 0.6 M NaCl, and 1% Nonidet P-40 (lane 3), and the soluble fractions were analyzed. The remaining cell pellets were solubilized in 1% SDS (lane 4). The position of the At-P4H-1 is indicated by an arrow.

A cDNA encoding At-P4H-1 residues Ser²³-Ser²⁸³ was also cloned into the pET15b E. coli expression vector with an N-terminal histidine tag. The recombinant polypeptide expressed in E. coli remained insoluble, however, and accumulated into inclusion bodies (data not shown).

The Recombinant At-P4H-1 Is a P4H That Hydroxylates Poly(L-proline)-- To study whether the recombinant A. thaliana polypeptide expressed in insect cells had any P4H activity, 50 µl of the soluble fraction of the cell homogenate was assayed by a method based on the hydroxylation-coupled decarboxylation of 2-oxo-[1-¹⁴C]glutarate (32). When 0.1 mg/ml poly(L-proline) (M_r 5,000) was used as the peptide substrate, a significant amount of P4H activity was observed even in the sample homogenized in the buffer containing 0.1% Triton X-100 (typically approximately 7000 cpm over various background values of approximately 200-300 cpm), although the recombinant enzyme could not be readily detected in the soluble fraction when analyzed by Coomassie Blue-stained SDS-PAGE (Fig. 2, lane 1). A further increase in the amount of activity was observed when the polypeptide was solubilized more efficiently, the activity levels ranging up to more than 30,000 cpm (an example is shown in Table II). Gel filtration experiments in a calibrated HiPrep Sephacryl S-100 HR column showed that enzyme activity was eluted in fractions that corresponded to a molecular weight of approximately 30,000 (details not shown). As the calculated molecular weight of the recombinant At-P4H-1 without the signal peptide is 29,252, the recombinant At-P4H-1 is a monomer.

As expected, the A. thaliana P4H required Fe²⁺, 2-oxoglutarate, O₂, and ascorbate (details not shown). The K_m for Fe²⁺ (Table I) was close to the values reported for partially purified P4Hs from the algae Chlamydomonas reinhardtii and Volvox carteri (21, 22), but approximately 40-fold higher than that of the PBCV-1 enzyme (26). The K_m for 2-oxoglutarate (Table I) was between those of the algal enzymes (21, 22), but approximately 6-fold higher than those of the PBCV-1 (26) and human enzymes (6, 34) and approximately the same as those of lysyl hydroxylase isoenzymes 1 and 3 (37, 38). The K_m for ascorbate was approximately the same (Table I) as those of the algal, PBCV-1, and human P4Hs (6, 21, 22, 26, 34). The K_m for poly(L-proline), M_r 5,000, was 5-10-fold lower (Table I) than for poly(L-proline), M_r 7,000, with algal enzymes (21, 22), and that for poly(L-proline), M_r 20,000, was 35-fold lower than that for poly(L-proline), M_r 31,000, with an algal enzyme (21), and 2500-fold lower than that for poly(L-proline), M_r 13,000, with the viral enzyme (26). K_m values ranging from 4 to 40 µM and a value of 5 µM have previously been reported for poly(L-proline), M_r 6,000 and 30,000, respectively, in the case of P4Hs from higher plants (23, 39-41).

Histidines 180 and 260, Aspartate 182, Lysine 270, and Arginine 278 are the Catalytically Critical Residues in At-P4H-1-- A sequence homology comparison (Fig. 1) indicated that the At-P4H-1 residues His¹⁸⁰, Asp¹⁸², and His²⁶⁰ correspond to the Fe²⁺ binding residues in the human P4H (I) subunit, the At-P4H-1 Lys²⁷⁰ corresponds to the lysine that binds the C-5 carboxyl group of 2-oxoglutarate, and the At-P4H-1 Arg²⁷⁸ corresponds to the fifth critical residue, a histidine or an arginine depending on the species, which is probably involved in both the binding of the C-1 carboxyl group of 2-oxoglutarate to the Fe²⁺ atom and the decarboxylation of this cosubstrate (12, 26, 34). To study the function of these residues in the At-P4H-1 polypeptide, His¹⁸⁰, Asp¹⁸², and His²⁶⁰ were converted individually to alanine and glutamate, Lys²⁷⁰ to alanine and arginine, and Arg²⁷⁸ to alanine and histidine. The crystal structure of cephalosporin synthase (42) has shown that, in addition to forming a salt bridge with an arginine residue (that corresponds to the lysine in P4Hs), the C-5 carboxyl group of 2-oxoglutarate is hydrogen-bonded to a serine residue in position +2 with respect to the positively charged arginine. We therefore also studied the role of Ser²⁷² in the catalytic activity of At-P4H-1 by converting it to alanine.

The mutant At-P4H-1 polypeptides were expressed in insect cells, and the cells were harvested, homogenized, and assayed for P4H activity as above. Mutation of the Fe²⁺ binding residues His¹⁸⁰, Asp¹⁸², or His²⁶⁰ to alanine or glutamate completely inactivated At-P4H-1 (Table II). Mutation of Lys²⁷⁰ to alanine or arginine also inactivated the enzyme completely, whereas mutation of Ser²⁷² to alanine reduced the enzyme activity by 83% (Table II). Conversion of the At-P4H-1 residue Arg²⁷⁸ to alanine completely inactivated the enzyme, whereas its replacement with a histidine reduced the activity to approximately 26% (Table II).

The A. thaliana P4H Hydroxylates Peptides Corresponding to Proline-rich Sequences Coded by Its Genome and Other Proline-rich Peptides-- The A. thaliana genome codes for extensins and arabinogalactan proteins that are known to be rich in 4-hydroxyproline (17-20). The synthetic peptides (Ala-Thr-Pro-Pro-Pro-Val)₃, representing arabinogalactan protein (GenBank^TM accession no. AAC77826), and Ser-Pro-Pro-Pro-Pro-Val-Ser-Pro-Pro-Pro-Val-Ser-Pro-Pro-Pro-Pro-Val and Ser-Pro-Pro-Pro-Val-Tyr-Lys-Ser-Pro-Pro-Pro-Pro-Val-Lys-His-Tyr-Ser-Pro-Pro-Pro-Val-Tyr-Lys, representing extensin (GenBank^TM accession no. S71227), were therefore tested as substrates for At-P4H-1. All these peptides were found to serve as substrates, their K_m values ranging from 10 to 40 µM (Table III). In addition, the synthetic peptides (Pro-Ala-Pro-Lys)₃, (Pro-Ala-Pro-Lys)_10, (Ser-Pro-Lys-Pro-Pro)₅, and (Pro-Glu-Pro-Pro-Ala)₅, representing sequences coded by the PBCV-1 genome and known to function as substrates for the recombinant viral P4H (26), served as substrates, with K_m values ranging from 2 to 90 µM (Table III). The K_m for (Ser-Pro-Lys-Pro-Pro)₅ was identical to that with the PBCV-1 enzyme, whereas those for (Pro-Ala-Pro-Lys)₃ and (Pro-Ala-Pro-Lys)₁₀ were approximately 3- and 10-fold lower than the corresponding values for the viral enzyme and that for (Pro-Glu-Pro-Pro-Ala)₅ 250-fold lower (26).

The A. thaliana Enzyme Effectively Hydroxylates Collagen-like Peptides Acting Preferentially on Prolines Preceding Glycine-- Highly surprisingly, the A. thaliana enzyme was found to hydroxylate denatured (Pro-Pro-Gly)₁₀ with a K_m of approximately 60 µM (Table IV), this value being similar to the K_m values of 20 and 100 µM determined for human type I and type II prolyl 4-hydroxylases, respectively (6, 34). The V_max of At-P4H-1 with (Pro-Pro-Gly)₁₀ was close to that obtained with poly(L-proline) (Table IV). The K_m values for (Pro-Pro-Gly)₅ and (Ala-Pro-Gly)₅ were approximately 120 and 100 µM, the V_max values with these peptides being 65 and 50% of that obtained with (Pro-Pro-Gly)₁₀.

To study the hydroxylation pattern of the (Pro-Pro-Gly)₁₀ peptide, it was partially hydroxylated with the recombinant At-P4H-1, purified from the reaction mixture by anion exchange chromatography and HPLC, and subjected to N-terminal sequencing. The Y position prolines in the repeating X-Pro-Gly triplets were found to be preferentially hydroxylated (Fig. 3), and the hydroxylation pattern was similar to the pattern observed with vertebrate P4Hs in that the Y position proline in the 9th triplet from the N terminus was most readily hydroxylated (43, 44). The sequencing results also indicated the presence of small amounts of 4-hydroxyproline in the X positions. The values shown in Fig. 3 have been corrected for the amounts of proline and 4-hydroxyproline carried over from the previous sequencing cycles but not for a significant background, as it could not be quantitated accurately. Therefore, the true degrees of hydroxylation of proline residues in the X positions are even smaller than those shown in Fig. 3. The data obtained in three additional experiments were essentially identical to those shown.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Analysis of the hydroxylation of the proline residues in (Pro-Pro-Gly)10 by At-P4H-1. The hydroxylation reaction was carried out with 100 µg/ml (Pro-Pro-Gly)10 as the substrate in the standard P4H reaction mixture under conditions that gave a high extent of hydroxylation of the substrate but not complete hydroxylation. The peptide was purified from the reaction mixture with HiTrap Q-Sepharose and HPLC and subjected to N-terminal sequencing. The columns indicate the degree of hydroxylation of the proline residues in the X and Y positions in the X-Y-Gly triplets. The values have been corrected for the amounts of proline and 4-hydroxyproline carried over from the previous sequencing cycles but not for a significant background, as it could not be quantitated accurately. P, proline; G, glycine.

To study the hydroxylation of proline residues in X positions further, the peptides (Gly-Pro-4Hyp)₅ and (Pro-Ala-Gly)₅ were tested as substrates (Table IV). The K_m value for (Pro-Ala-Gly)₅ was found to be 280 µM, and the V_max values obtained with it and (Gly-Pro-4Hyp)₅ were 20% and less than 5% of that obtained with (Pro-Pro-Gly)₁₀, respectively (Table IV). Because of the low reaction rate, the K_m for (Gly-Pro-4Hyp)₅ could not be determined. Measurement of the amount of 4-hydroxyproline formed in a 1-ml P4H reaction mixture containing 200 µM (Pro-Ala-Gly)₅ by a colorimetric method showed this amount to be approximately 30% of that formed in the (Ala-Pro-Gly)₅ peptide in the same experiment (details not shown). This percentage is lower than the V_max of 40% for (Pro-Ala-Gly)₅ relative to (Ala-Pro-Gly)₅ (Table IV), evidently because of the combined effect of the higher K_m and lower V_max.

The A. thaliana Enzyme Effectively Hydroxylates Peptides Representing Transcription Factor HIF Sequences and Containing Only One Proline Residue-- The recombinant At-P4H-1 was also found to effectively hydroxylate synthetic peptides representing the two hydroxylated sequences in human transcription factor HIF-1 (13-16). The K_m values for the peptides representing the N-terminal (Asp-Ala-Leu-Thr-Leu-Leu-Ala-Pro-Ala-Ala-Gly-Asp-Thr-Ile-Ile-Ser-Leu-Phe-Gly) and Cterminal (Asp-Leu-Asp-Leu-Glu-Met-Leu-Ala-Pro-Tyr-Ile-Pro-MetAsp-Asp-Asp-Phe-Gln-Leu) hydroxylation sites in HIF were 100 and 50 µM, respectively (Table V). The V_max with the peptide representing the N-terminal hydroxylation site was approximately 70% of that obtained with poly(L-proline), whereas that with the peptide representing the C-terminal hydroxylation site was approximately 20% (Table V).

View this table: [in this window] [in a new window]   Table V Km and Vmax values of At-P4H-1 for HIF peptides Km and Vmax values were determined as described previously (34).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The data reported here indicate that the A. thaliana genome probably encodes a family of P4Hs with at least six members. The high degree of sequence similarity and the strict conservation of the residues that bind the Fe²⁺ atom and the C-5 carboxyl group of 2-oxoglutarate suggest that all six are P4Hs. The animal P4Hs had for a long time been assumed to be of one type only, until a second isoenzyme was cloned and characterized from mouse and human tissues (5, 6), and very recently a family of three additional cytoplasmic human P4Hs has been cloned and shown to be involved in the hydroxylation of the hypoxia-inducible transcription factor HIF (15, 16). Our searches of the completed genomes of C. elegans and D. melanogaster suggest that the former may have more than 5 P4Hs and the latter more than 10, although only 4 P4Hs have so far been cloned from the former (7, 9-11, 15) and 2 from the latter (12, 16). P4Hs thus appear to constitute enzyme families in both plant and animal tissues.

The At-P4H-1 cloned and characterized here was found to be a 29-kDa monomer. Studies of partially purified P4Hs from unicellular and multicellular green algae have likewise indicated that these enzymes are monomers (21, 22), whereas early studies of a P4H from Phaseolus vulgaris suggested that this enzyme may have two kinds of subunit (23). Subsequent work has demonstrated, however, that the ratio of the co-purifying polypeptide to the catalytic polypeptide varies in a range well below 1:1 (24), indicating that this P4H is likewise a monomer. It thus seems that plant P4Hs, like the animal P4Hs involved in the hydroxylation of HIF (15, 16), may be monomers, whereas the vertebrate P4Hs involved in the hydroxylation of collagens are ₂₂ tetramers (3, 4).

Site-directed mutagenesis showed that replacement of the conserved At-P4H-1 residues His¹⁸⁰, Asp¹⁸², and His²⁶⁰, which correspond to the Fe²⁺-binding ligands in human P4H (34), by alanine or glutamate completely inactivated the enzyme, thus demonstrating their critical role in catalytic activity. The corresponding mutations of the Fe²⁺-binding residues also result in complete inactivation in human type I P4H, with the exception that replacement of Asp⁴¹⁴ with glutamate retains approximately 15% of the activity (34). These results differ from those with aspartyl (asparaginyl) -hydroxylase, in which replacement of the Fe²⁺-binding histidines with a negatively charged amino acid resulted in 10-20% residual activity (45). Mutation of At-P4H-1 Lys²⁷⁰, which corresponds to the Lys⁴⁹³ that ionically binds the C-5 carboxyl group of 2-oxoglutarate in human type I P4H (34), to alanine or arginine completely inactivated the enzyme. In comparison, replacement of Lys⁴⁹³ in human type I P4H with arginine (34), and the corresponding Arg⁷⁰⁰ in lysyl hydroxylase 1 with lysine (46), reduced the activities to approximately 15%, whereas replacement with alanine completely inactivated the enzymes. In the case of cephalosporin synthase, the C-5 carboxyl group of 2-oxoglutarate forms a salt bridge to an arginine and a hydrogen bond to a serine in position +2 with respect to the arginine (42). The critical role of the corresponding serine in At-P4H-1 was demonstrated here by 83% inactivation of the enzyme when Ser²⁷² was converted to alanine. An additional catalytically important positively charged residue in animal P4Hs is located in position +8 with respect to the lysine that binds the C-5 carboxyl group of 2-oxoglutarate (34). This residue is probably involved in both the binding of the C-1 carboxyl group of 2-oxoglutarate and the decarboxylation of this cosubstrate (34). Replacement of this His⁵⁰¹ in human type I P4H with a serine completely inactivates the enzyme, whereas substitution with arginine or lysine reduces the activity to approximately 10-15% (34). Likewise, replacement of the corresponding Arg²⁷⁸ in At-P4H-1 with alanine completely inactivated the enzyme, whereas mutation to histidine reduced the activity to approximately 26%. The corresponding mutations Arg⁴⁹⁰  Ser and Arg⁴⁹⁰  His inactivated a Drosophila P4H by 70 and 10%, respectively (12).

The most distinct difference in catalytic properties between plant and animal P4Hs is found in the hydroxylation of poly(L-proline). This polypeptide is an effective substrate for all plant P4Hs studied (21-25, 39-41), whereas some animal P4Hs recognize it as an effective competitive inhibitor and some as a weak one, but none of the animal P4Hs characterized so far has used it as a substrate (3, 4). Data indicating that free proline, (Pro)₂, and (Pro)₃ are not hydroxylated by the P4H from Vinca rosea have been interpreted as indicating that plant P4Hs may require a poly(L-proline) type II helix conformation (39, 47). The recombinant viral PBCV-1 P4H likewise used poly(L-proline) as an efficient substrate (26). In the present work recombinant At-P4H-1 efficiently hydroxylated poly(L-proline), the K_m values for M_r 5000 and 20,000 poly(L-prolines) being 2 and 0.2 µM, respectively, i.e. lower than those previously reported for any algal or higher plant P4Hs.

The recombinant At-P4H-1 also efficiently hydroxylated other proline-rich peptides, the K_m values for peptides representing A. thaliana arabinogalactan protein and extensin sequences varying between 10 and 40 µM. Synthetic peptides representing proline-rich sequences coded by the PBCV-1 genome were likewise efficiently hydroxylated, with K_m values varying from 2 to 90 µM. Highly interestingly, the recombinant At-P4H-1 also hydroxylated peptides representing the two hydroxylated sequences in the human HIF, with K_m values of 100 and 50 µM, respectively. Unlike the HIF P4Hs (15), At-P4H-1 hydroxylated the peptide representing the N-terminal hydroxylation site in HIF at a higher rate, the V_max obtained with this peptide being approximately 3.5-fold when compared with that obtained with the peptide representing the C-terminal hydroxylation site. As only one proline residue is present in each of the HIF peptides, these data clearly indicate that a poly(L-proline) type II helix conformation is not required for hydroxylation by At-P4H-1. A similar conclusion on sequence-specific rather than poly(L-proline) II conformation-specific hydroxylation by plant P4Hs was recently reached in a comparison of sequences containing 4-hydroxyproline present in various plant proteins (48).

A highly surprising finding was that the recombinant A. thaliana P4H also effectively hydroxylated (Pro-Pro-Gly)₁₀, with a K_m similar to those reported for vertebrate collagen P4Hs (3-6, 34, 36) and a V_max close to that obtained with poly(L-proline). Plant P4Hs have previously been reported either not to hydroxylate (Pro-Pro-Gly)₁₀ at all or to hydroxylate it only at a very low rate (21, 22, 39-41). Although an early study indicated that a partially purified carrot P4H hydroxylates protocollagen, a protein consisting of nonhydroxylated procollagen chains (49), subsequent efforts have been unable to validate these results (25). The viral PBCV-1 P4H that hydroxylates poly(L-proline) was also found to hydroxylate (Pro-Pro-Gly)₁₀, but with a K_m of approximately 2.9 mM, i.e. approximately 50-fold higher than the K_m of 60 µM measured here for At-P4H-1. Sequencing of a (Pro-Pro-Gly)₁₀ peptide partially hydroxylated by the recombinant At-P4H-1 showed that the enzyme had acted preferentially on prolines preceding glycine. However, unlike the animal P4Hs, At-P4H-1 was not absolutely specific for hydroxylation of only these positions, as small amounts of 4-hydroxyproline were also found in the positions following glycine. In agreement with this, (Gly-Pro-4Hyp)₅ was found to serve as a substrate, although the V_max obtained with it was less than 5% of that obtained with (Pro-Pro-Gly)₁₀. Preferential hydroxylation of proline in the Y position of X-Y-Gly triplets was also seen in a comparison of the hydroxylation of (Ala-Pro-Gly)₅ and (Pro-Ala-Gly)₅, in that the K_m of the latter was approximately 3-fold and the V_max approximately 40%. It may be noted that (Pro-Ala-Gly)_n has also been reported to be hydroxylated by a vertebrate P4H in vitro, although at a rate that is only approximately 7% of that obtained with (Ala-Pro-Gly)_n (50), and the tetrapeptide Pro-Pro-Ala-Pro is likewise known to act as a substrate for vertebrate P4Hs because of hydroxylation of the proline preceding alanine (51). Interestingly, the pattern of hydroxylation of the Y position prolines in (Pro-Pro-Gly)₁₀ with At-P4H-1 was similar to the asymmetrical hydroxylation pattern produced by the vertebrate enzymes, the 9th triplet from the N-terminal end being most readily hydroxylated (43, 44), whereas no asymmetric hydroxylation was seen with the viral PBCV-1 enzyme (26).

	ACKNOWLEDGEMENTS

We thank Hongmin Tu for performing the amino acid analyses and N-terminal sequencing and Merja Nissilä, Eeva Lehtimäki, Raija Juntunen, Tanja Väisänen, and Liisa Äijälä for expert technical assistance.

	FOOTNOTES

^* This work was supported by a Health Science Council grant and by Grant 44843 from the Finnish Centre of Excellence Programme 2000-2005 of the Academy of Finland.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Medical Biochemistry and Molecular Biology, P. O. Box 5000, University of Oulu, FIN-90014 Oulu, Finland. Tel.: 358-8-537-5740; Fax: 358-8-537-5811; E-mail: johanna.myllyharju@oulu.fi.

Published, JBC Papers in Press, April 25, 2002, DOI 10.1074/jbc.M201865200

	ABBREVIATIONS

The abbreviations used are: P4H, prolyl 4-hydroxylase; HIF, hypoxia-inducible transcription factor; PBCV-1, P. bursaria chlorella virus-1; HPLC, high performance liquid chromatography; DTT, dithiothreitol.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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