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• W2010060668 abstract "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 α 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)10and 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 μm. 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. 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 α 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)10and 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 μm. 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. 4-Hydroxyproline is the characteristic amino acid of collagens and more than 10 other animal proteins with collagen-like sequences. This amino acid plays a central role in all collagens, as the hydroxy groups of the 4-hydroxyproline residues are essential for the formation of the collagen triple helix at body temperature. 4-Hydroxyproline is also found in certain proline-rich plant proteins, but it has been believed to be absent from viral and bacterial proteins (for reviews, see Refs.1Showalter A.M. Varner J.E. Marcus A. The Biochemistry of Plants: A Comprehensive Treatise. 15. Academic Press, New York1989: 485-520Google Scholar, 2Kivirikko K.I. Myllylä R. Pihlajaniemi T. Harding J.J. Crabbe M.J.C. Post-Translational Modifications of Proteins. CRC Press, Boca Raton, FL1992: 1-51Google Scholar, 3Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1355) Google Scholar, 4Sommer-Knudsen J. Bacic A. Clarke A.E. Phytochemistry. 1998; 47: 483-497Crossref Scopus (133) Google Scholar). The formation of 4-hydroxyproline is catalyzed by prolyl 4-hydroxylases that act on proline residues in peptide linkages. The vertebrate enzymes are 240-kDa α2β2 tetramers, in which the catalytic sites are located in the α subunits and the β subunits are identical to the enzyme and chaperone protein disulfide isomerase. They require Fe2+, 2-oxoglutarate, O2, and ascorbate and hydroxylate -X-Pro-Gly- sequences (for reviews, see Refs. 5Kivirikko K.I. Myllyharju J. Matrix Biol. 1998; 16: 357-368Crossref PubMed Scopus (233) Google Scholar and 6Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Related Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar). Prolyl 4-hydroxylases from higher plants may resemble the vertebrate enzymes in their structure (7Bolwell G.P. Robbins M.P. Dixon R.A. Eur. J. Biochem. 1985; 148: 571-578Crossref PubMed Scopus (95) Google Scholar), whereas prolyl 4-hydroxylases from multicellular and unicellular green algae are 60-kDa monomers (8Kaska D.D. Günzler V. Kivirikko K.I. Myllylä R. Biochem. J. 1987; 241: 483-490Crossref PubMed Scopus (31) Google Scholar, 9Kaska D.D. Myllylä R. Günzler V. Gibor A. Kivirikko K.I. Biochem. J. 1988; 256: 257-263Crossref PubMed Scopus (24) Google Scholar). Plant prolyl 4-hydroxylases require the same cosubstrates as the animal enzymes, but they differ from the latter in that they hydroxylate proline residues in poly(l-proline) and poly(l-proline)-like sequences, while the repeating -X-Pro-Gly- triplets are either very poor substrates or not hydroxylated at all (2Kivirikko K.I. Myllylä R. Pihlajaniemi T. Harding J.J. Crabbe M.J.C. Post-Translational Modifications of Proteins. CRC Press, Boca Raton, FL1992: 1-51Google Scholar, 8Kaska D.D. Günzler V. Kivirikko K.I. Myllylä R. Biochem. J. 1987; 241: 483-490Crossref PubMed Scopus (31) Google Scholar). We report here that the genome of Paramecium bursaria Chlorella virus-1 (PBCV-1 1The abbreviations used are: PBCV-1, Paramecium bursaria Chlorella virus-1; PCR, polymerase chain reaction; IPTG, isopropyl-β-d-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography 1The abbreviations used are: PBCV-1, Paramecium bursaria Chlorella virus-1; PCR, polymerase chain reaction; IPTG, isopropyl-β-d-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; Refs. 10Lu Z. Li Y. Zhang Y. Kutish G.F. Rock D.L. Van Etten J.L. Virol. 1995; 206: 339-352Crossref PubMed Scopus (62) Google Scholar and 11Li Y. Lu Z. Burbank D.E. Kutish G.F. Rock D.L. Van Etten J.L. Virol. 1995; 212: 134-150Crossref PubMed Scopus (49) Google Scholar) encodes a 242-amino acid polypeptide that shows a distinct amino acid sequence similarity to the C-terminal half of the catalytic α subunits of animal prolyl 4-hydroxylases. In addition, the genome contains many open reading frames for proteins with proline-rich repeats. The recombinant viral polypeptide, expressed inEscherichia coli, was found to be a soluble monomer and to hydroxylate (Pro-Pro-Gly)10, poly(l-proline), and several synthetic peptides corresponding to proline-rich repeats coded by the viral genome. The data strongly suggest that PBCV-1 expresses proteins in which a number of proline residues become hydroxylated by a viral prolyl 4-hydroxylase with many unique properties. Thus the occurrence of 4-hydroxyproline in proteins is probably not restricted to certain animal and plant proteins. A sequence homology search in GenBankTMusing The Basic Local Alignment Search Tool (12Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (58771) Google Scholar) indicated the presence in the PBCV-1 genome (accession number U42580) of an open reading frame encoding a 242-amino acid polypeptide that showed a similarity to the C-terminal half of the human prolyl 4-hydroxylase α(I) subunit (13Helaakoski T. Vuori K. Myllylä R. Kivirikko K.I. Pihlajaniemi T. Proc. Natl Acad. Sci. U. S. A. 1989; 86: 4392-4396Crossref PubMed Scopus (103) Google Scholar). This amino acid sequence was aligned with those of the α(I) and α(II) subunits of human type I and type II prolyl 4-hydroxylases (13Helaakoski T. Vuori K. Myllylä R. Kivirikko K.I. Pihlajaniemi T. Proc. Natl Acad. Sci. U. S. A. 1989; 86: 4392-4396Crossref PubMed Scopus (103) Google Scholar,14Annunen P. Helaakoski T. Myllyharju J. Veijola J. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1997; 272: 17342-17348Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and the α subunits of the Caenorhabditis elegans (15Veijola J. Koivunen P. Annunen P. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1994; 269: 26746-26753Abstract Full Text PDF PubMed Google Scholar) and Drosophila melanogaster (16Annunen P. Koivunen P. Kivirikko K.I. J. Biol. Chem. 1999; 274: 6790-6796Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) prolyl 4-hydroxylases by the ClustalW method (17Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (54908) Google Scholar). The cleavage site of the signal peptide was predicted using the computational parameters of von Hejne (18Von Hejne G. Nucleic Acids Res. 1986; 14: 4683-4690Crossref PubMed Scopus (3685) Google Scholar). PCR primers 5′-CGCGCATATGGAGGGGTTTGAAACCAGCGAT-3′ and 5′-CGCGCTCGAGTCATTTAACAGCACGGATCCATT-3′ were synthesized based on the viral DNA sequence and used to obtain a 621-base pair PCR product flanked by NdeI and XhoI restriction sites from the viral genomic DNA. This PCR product coding for the amino acids Glu-36–Lys-242 of the viral prolyl 4-hydroxylase-like polypeptide was cloned to NdeI-XhoI-digested pET15b expression vector (Novagen), and the sequence was verified in an automated DNA sequencer (Applied Biosystems). The expression plasmid was transformed into the E. coliBL21(DE3) strain (Novagen). The cells were grown at 37 °C to an optical density of 0.55 at 600 nm, incubated at 28 °C for 30 min, and expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to 0.8 mm. The cells were harvested 3 h after induction, suspended in a 0.05 volume of a solution of 5 mm imidazole, 0.5 m NaCl, and 20 mm Tris, pH 7.9, sonicated until the sample was no longer viscous, centrifuged at 38,000 ×g for 30 min, and the soluble and insoluble fractions were analyzed by 12% SDS-PAGE. The recombinant PBCV-1 polypeptide was purified by applying the soluble fraction of the cell lysate to a Ni2+-chelate affinity column (Invitrogen); the unbound material was removed by washing with a solution of 60 mmimidazole, 0.5 m NaCl, and 20 mm Tris, pH 7.9; and the recombinant polypeptide was eluted by increasing the imidazole concentration to 0.5 m. The fractions were analyzed by 12% SDS-PAGE and those containing the polypeptide were pooled and concentrated with Macrosep 10K concentrators (Filtron). The apparent molecular weight of the purified protein was estimated by applying it to a calibrated HiLoad 16/60 Superdex S-200 (Amersham Pharmacia Biotech) column, equilibrated, and eluted with a 0.3 mNaCl, 50 mm sodium phosphate buffer, pH 7.0. Prolyl 4-hydroxylase activity was assayed by a method based on the hydroxylation-coupled decarboxylation of 2-oxo-[1-14C]glutarate (19Kivirikko K.I. Myllylä R. Methods Enzymol. 1982; 82: 245-304Crossref PubMed Scopus (320) Google Scholar). In some experiments the (Pro-Ala-Pro-Lys)5 substrate was purified from the reaction mixture by reverse phase HPLC, hydrolyzed using the manual gas-phase hydrolysis method, and analyzed in an Applied Biosystems 421A amino acid analyzer. N-terminal sequencing of the purified (Pro-Ala-Pro-Lys)5 peptide was performed in an Applied Biosystems 477A pulse-liquid protein sequencer. K mand V max values were determined as described previously (20Myllyharju J. Kivirikko K.I. EMBO J. 1997; 16: 1173-1180Crossref PubMed Scopus (162) Google Scholar). A sequence homology search indicated that the genome of PBCV-1 (Refs. 10Lu Z. Li Y. Zhang Y. Kutish G.F. Rock D.L. Van Etten J.L. Virol. 1995; 206: 339-352Crossref PubMed Scopus (62) Google Scholar and 11Li Y. Lu Z. Burbank D.E. Kutish G.F. Rock D.L. Van Etten J.L. Virol. 1995; 212: 134-150Crossref PubMed Scopus (49) Google Scholar; GenBankTM accession numberU42580) contains an open reading frame encoding a 242-amino acid polypeptide that shows a distinct sequence similarity to the C-terminal half of the catalytic α subunits of prolyl 4-hydroxylases from various animal sources (Fig. 1). A putative signal sequence is located at its N terminus, the most likely first amino acid of the processed viral polypeptide being glutamate (Fig. 1), based on the computational parameters of von Hejne (18Von Hejne G. Nucleic Acids Res. 1986; 14: 4683-4690Crossref PubMed Scopus (3685) Google Scholar). Thus the length of the signal peptide is probably 32 residues and that of the processed polypeptide 210 amino acids. The sequence of the processed viral polypeptide is 20% identical to residues 294–504 in the 517-residue α subunit of human type I prolyl 4-hydroxylase (13Helaakoski T. Vuori K. Myllylä R. Kivirikko K.I. Pihlajaniemi T. Proc. Natl Acad. Sci. U. S. A. 1989; 86: 4392-4396Crossref PubMed Scopus (103) Google Scholar) and 15–23% identical to the corresponding residues in the α subunits of the human type II prolyl 4-hydroxylase (14Annunen P. Helaakoski T. Myllyharju J. Veijola J. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1997; 272: 17342-17348Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and theC. elegans (15Veijola J. Koivunen P. Annunen P. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1994; 269: 26746-26753Abstract Full Text PDF PubMed Google Scholar) and D. melanogaster (16Annunen P. Koivunen P. Kivirikko K.I. J. Biol. Chem. 1999; 274: 6790-6796Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) prolyl 4-hydroxylases (Fig. 1). The two histidines and one aspartate that bind the Fe2+ atom at the catalytic site (20Myllyharju J. Kivirikko K.I. EMBO J. 1997; 16: 1173-1180Crossref PubMed Scopus (162) Google Scholar, 21Myllylä R. Günzler V. Kivirikko K.I. Kaska D.D. Biochem. J. 1992; 286: 923-927Crossref PubMed Scopus (57) Google Scholar, 22Lamberg A. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1995; 270: 9926-9931Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and the lysine that binds the C-5 carboxyl group of the 2-oxoglutarate (20Myllyharju J. Kivirikko K.I. EMBO J. 1997; 16: 1173-1180Crossref PubMed Scopus (162) Google Scholar) are all conserved in the PBCV-1 sequence (His-152, Asp-154, His-221, and Lys-231 in Fig. 1). Since the last mentioned residue in all other 2-oxoglutarate dioxygenases, including the closely related enzyme lysyl hydroxylase (23Passoja K. Myllyharju J. Pirskanen A. Kivirikko K.I. FEBS Lett. 1998; 434: 145-148Crossref PubMed Scopus (32) Google Scholar), is an arginine (21Myllylä R. Günzler V. Kivirikko K.I. Kaska D.D. Biochem. J. 1992; 286: 923-927Crossref PubMed Scopus (57) Google Scholar, 24Roach P.L. Clifton I.J. Fülöp V. Harlos K. Barton G.J. Hajdu J. Andersson I. Schofield C.J. Baldwin J.E. Nature. 1995; 375: 700-704Crossref PubMed Scopus (377) Google Scholar, 25Lukacin R. Britsch L. Eur. J. Biochem. 1997; 249: 748-757Crossref PubMed Scopus (120) Google Scholar), we regarded it as possible that the viral polypeptide might be a prolyl 4-hydroxylase. The fifth critical residue at the catalytic site of the vertebrate prolyl 4-hydroxylases, a histidine that is probably involved in the binding of the C-1 carboxyl group of 2-oxoglutarate to the Fe2+ atom and in the decarboxylation of this cosubstrate (20Myllyharju J. Kivirikko K.I. EMBO J. 1997; 16: 1173-1180Crossref PubMed Scopus (162) Google Scholar), is replaced in the PBCV-1 sequence as in theDrosophila α subunit sequence by an arginine (Arg-239 in Fig. 1). However, the PBCV-1 sequence shows no similarity to the peptide substrate binding domain present between residues 140–240 in the α subunits of animal prolyl 4-hydroxylases (26Myllyharju J. Kivirikko K.I. EMBO J. 1999; 18: 306-312Crossref PubMed Scopus (60) Google Scholar). To express the viral polypeptide in E. coli, the PBCV-1 DNA sequence coding for amino acids Glu-36–Lys-242 was synthesized by PCR, cloned into the pET-15b vector with an N-terminal histidine tag, and transformed into the BL21(DE3) host strain. Expression of the polypeptide was induced with IPTG, and the cells were incubated at 28 °C for 3 h. The cells were then harvested, suspended in a Tris-HCl buffer, pH 7.9, containing 5 mm imidazole, sonicated, and the soluble and insoluble fractions were analyzed by 12% SDS-PAGE and Coomassie Blue staining (Fig. 2, lanes 2 and3). The expressed recombinant polypeptide was mainly found in the soluble fraction (Fig. 2, lane 2) and could be purified using a Ni2+-chelate affinity column and imidazole elution (Fig. 2, lane 4). Gel filtration in a calibrated Superdex S-200 column indicated that the recombinant polypeptide had an apparent molecular weight of about 30,000 (details not shown). As the calculated molecular weight of the recombinant polypeptide with the N-terminal histidine tag and the thrombin cleavage site is 27,195, the recombinant polypeptide was apparently a monomer. To study whether the viral polypeptide had any prolyl 4-hydroxylase activity, 10 μg of the purified protein was assayed as a possible enzyme by a method based on the hydroxylation-coupled decarboxylation of 2-oxo-[1-14C]glutarate (19Kivirikko K.I. Myllylä R. Methods Enzymol. 1982; 82: 245-304Crossref PubMed Scopus (320) Google Scholar). When 0.5 mg/ml of (Pro-Pro-Gly)10 was used as the peptide substrate, the amount of 14CO2 generated was 5450 cpm, whereas various negative controls gave less than 500 cpm. Poly(l-proline), M r 40,000, a competitive inhibitor of animal prolyl 4-hydroxylases (5Kivirikko K.I. Myllyharju J. Matrix Biol. 1998; 16: 357-368Crossref PubMed Scopus (233) Google Scholar, 6Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Related Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar), also acted as a substrate, giving 5850 cpm under the above conditions. The pH optimum of the hydroxylation reaction was 7.0 (details not shown). The viral enzyme, like the animal and plant prolyl 4-hydroxylases, required Fe2+, 2-oxoglutarate, O2, and ascorbate (details not shown). The K m values for the cosubstrates Fe2+, 2-oxoglutarate, and ascorbate were very similar to those of human type I prolyl 4-hydroxylase (TableI), suggesting that the cofactor binding sites of these enzymes may be similar. However, theK m value of the viral enzyme for the peptide substrate (Pro-Pro-Gly)10 was about 150-fold (Table I), and the K m values for poly(l-proline),M r 13,000 and 40,000 (Table I), were also much higher than those of 23 and 7 μm reported for poly(l-proline), M r 7,000 and 31,000, with the prolyl 4-hydroxylase from the unicellular green algaChlamydomonas reinhardii (8Kaska D.D. Günzler V. Kivirikko K.I. Myllylä R. Biochem. J. 1987; 241: 483-490Crossref PubMed Scopus (31) Google Scholar) or 10 μm for poly(l-proline), M r 7,000, with the prolyl 4-hydroxylase from the multicellular green alga Volvox carteri (9Kaska D.D. Myllylä R. Günzler V. Gibor A. Kivirikko K.I. Biochem. J. 1988; 256: 257-263Crossref PubMed Scopus (24) Google Scholar).Table IKm values of the PBCV-1 and human type I prolyl 4-hydroxylases for cosubstrates and for (Pro-Pro-Gly)10 and poly(l-proline)Cosubstrate or substrateK mPBCV-1Human type I prolyl 4-hydroxylaseaRef. 20.μmFe2+0.422-Oxoglutarate2020Ascorbate300300(Pro-Pro-Gly)10290020Poly(l-proline), M r = 13,000500cIbcI, competitive inhibitor.Poly(l-proline),M r = 40,000100cIbcI, competitive inhibitor.K m values were determined as described previously (20Myllyharju J. Kivirikko K.I. EMBO J. 1997; 16: 1173-1180Crossref PubMed Scopus (162) Google Scholar).a Ref. 20Myllyharju J. Kivirikko K.I. EMBO J. 1997; 16: 1173-1180Crossref PubMed Scopus (162) Google Scholar.b cI, competitive inhibitor. Open table in a new tab K m values were determined as described previously (20Myllyharju J. Kivirikko K.I. EMBO J. 1997; 16: 1173-1180Crossref PubMed Scopus (162) Google Scholar). The PBCV-1 genome contains many open reading frames coding for proline-rich repeats. These include (Pro-Ala-Pro-Lys)n, in which n is up to 26, (Ser-Pro-Lys-Pro-Pro)20, (Pro-Glu-Pro-Pro-Ala)9, (Ser-Thr-Lys-Pro-Pro)11, and (Glu-Pro-Ser-Pro-Glu-Pro)5. Synthetic peptides (Ser-Pro-Lys-Pro-Pro)5, (Pro-Glu-Pro-Pro-Ala)5, Lys-Pro-Ala, Pro-Ala-Pro-Lys, and (Pro-Ala-Pro-Lys)n, wheren = 2–10, were therefore tested as substrates for the recombinant PBCV-1 polypeptide. All these peptides were found to serve as substrates, their K m values ranging from 20 to 8600 μm (Table II). TheV max values for (Pro-Ala-Pro-Lys)n, where n = 3–10, were identical within the range of experimental error (Table II), and these values were also essentially identical to those for poly(l-proline),M r 13,000 and 40,000, and for (Pro-Pro-Gly)10 determined in the same experiments (details not shown), whereas the V max for (Pro-Ala-Pro-Lys)2 was about 40%, (Ser-Pro-Lys-Pro-Pro)5 15%, and those for (Pro-Glu-Pro-Pro-Ala)5, Pro-Ala-Pro-Lys, and Lys-Pro-Ala were even lower (Table II). Thus the best substrate among those tested when considering both K m andV max was (Pro-Ala-Pro-Lys)10. The generation of 4-hydroxyproline in the (Pro-Ala-Pro-Lys)5peptide was verified by amino acid analysis of the peptide purified from the hydroxylation reaction mixture by reverse phase HPLC (details not shown).Table IIKm and Vmax values of the PBCV-1 prolyl 4-hydroxylase for synthetic peptides corresponding to repeats coded by the viral genomeSubstrateK mV maxμmcpm/μg(Ser-Pro-Lys-Pro-Pro)5201800(Pro-Glu-Pro-Pro-Ala)51000400Lys-Pro-Ala8600200Pro-Ala-Pro-Lys4800400(Pro-Ala-Pro-Lys)29504600(Pro-Ala-Pro-Lys)331013,500(Pro-Ala-Pro-Lys)55010,300(Pro-Ala-Pro-Lys)102011,900K m values were determined as described previously (20Myllyharju J. Kivirikko K.I. EMBO J. 1997; 16: 1173-1180Crossref PubMed Scopus (162) Google Scholar). Open table in a new tab K m values were determined as described previously (20Myllyharju J. Kivirikko K.I. EMBO J. 1997; 16: 1173-1180Crossref PubMed Scopus (162) Google Scholar). The substrate requirements of the viral enzyme thus differed distinctly from those of both animal and plant prolyl 4-hydroxylases. The hydroxylation of (Pro-Pro-Gly)10 is a property similar to that of animal prolyl 4-hydroxylases. Although theK m of 2900 μm is much higher than theK m values of 20 and 100 μm of the human type I and type II enzymes (26Myllyharju J. Kivirikko K.I. EMBO J. 1999; 18: 306-312Crossref PubMed Scopus (60) Google Scholar), the Km of 20 μm of the C. elegans enzyme (27Veijola J. Annunen P. Koivunen P. Page A.P. Pihlajaniemi T. Kivirikko K.I. Biochem. J. 1996; 317: 721-729Crossref PubMed Scopus (31) Google Scholar) and 260 μm of the D. melanogaster enzyme (16Annunen P. Koivunen P. Kivirikko K.I. J. Biol. Chem. 1999; 274: 6790-6796Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), theV max of the viral enzyme for (Pro-Pro-Gly)10 was similar to itsV max values for poly(l-proline) and the best polypeptide substrates. Some plant prolyl 4-hydroxylases also hydroxylate (Pro-Pro-Gly)10, but only at a very low rate (8Kaska D.D. Günzler V. Kivirikko K.I. Myllylä R. Biochem. J. 1987; 241: 483-490Crossref PubMed Scopus (31) Google Scholar). The hydroxylation of poly(l-proline) is a property of plant prolyl 4-hydroxylases (2Kivirikko K.I. Myllylä R. Pihlajaniemi T. Harding J.J. Crabbe M.J.C. Post-Translational Modifications of Proteins. CRC Press, Boca Raton, FL1992: 1-51Google Scholar), whereas poly(l-proline) is a competitive inhibitor of the animal enzymes (6Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Related Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar), but theK m values of the viral enzyme for poly(l-proline) were more than 1 order of magnitude higher than those reported for plant enzymes (2Kivirikko K.I. Myllylä R. Pihlajaniemi T. Harding J.J. Crabbe M.J.C. Post-Translational Modifications of Proteins. CRC Press, Boca Raton, FL1992: 1-51Google Scholar, 8Kaska D.D. Günzler V. Kivirikko K.I. Myllylä R. Biochem. J. 1987; 241: 483-490Crossref PubMed Scopus (31) Google Scholar, 9Kaska D.D. Myllylä R. Günzler V. Gibor A. Kivirikko K.I. Biochem. J. 1988; 256: 257-263Crossref PubMed Scopus (24) Google Scholar). The best peptide substrates of the viral enzyme, (Pro-Ala-Pro-Lys)10 and (Ser-Pro-Lys-Pro-Pro)5, correspond to sequences coded by the viral genome. The K m values for the authentic viral polypeptides may be even lower, as the K mvalues decreased with an increase in the chain length of the substrates and as the actual viral repeat sequences range up to (Pro-Ala-Pro-Lys)26 and (Ser-Pro-Lys-Pro-Pro)20. In order to study whether prolines in both positions of the -Pro-Ala-Pro-Lys- repeat are hydroxylated, (Pro-Ala-Pro-Lys)5 was allowed to react with the viral prolyl 4-hydroxylase under conditions that gave a high extent but not complete hydroxylation. The peptide was then purified from the reaction mixture and subjected to amino acid sequencing. Prolines in both positions of the repeat were found to be hydroxylated, but those preceding alanines were hydroxylated more readily, except in the extreme N-terminal -Pro-Ala-Pro-Lys- repeat (Fig.3). The highest extents of hydroxylation were seen with prolines in the second and third repeat (Fig. 3). Interestingly, the pattern of hydroxylation of (Pro-Ala-Pro-Lys)5 with the viral prolyl 4-hydroxylase was found to be distinctly different from that of the hydroxylation of the 5 or 10 -Pro-Pro-Gly- triplets in (Pro-Pro-Gly)5 or (Pro-Pro-Gly)10 by the vertebrate enzyme (28Kivirikko K.I. Suga K. Kishida Y. Sakakibara S. Prockop D.J. Biochem. Biophys. Res. Commun. 1971; 45: 1591-1596Crossref PubMed Scopus (14) Google Scholar, 29Berg R.A. Kishida Y. Sakakibara S. Prockop D.J. Biochemistry. 1977; 16: 1615-1621Crossref PubMed Scopus (17) Google Scholar). The latter hydroxylates its substrates asymmetrically, so that the 4th or 9th triplet from the N-terminal end, respectively, is hydroxylated more readily than any other (28Kivirikko K.I. Suga K. Kishida Y. Sakakibara S. Prockop D.J. Biochem. Biophys. Res. Commun. 1971; 45: 1591-1596Crossref PubMed Scopus (14) Google Scholar, 29Berg R.A. Kishida Y. Sakakibara S. Prockop D.J. Biochemistry. 1977; 16: 1615-1621Crossref PubMed Scopus (17) Google Scholar), whereas no such asymmetric hydroxylation was seen with the viral enzyme (Fig. 3). The present data indicate that the genome of PBCV-1 encodes an active prolyl 4-hydroxylase with many unique properties and a number of protein sequences that can be hydroxylated by the enzyme. The unique properties of the enzyme include its low molecular weight and specificity with respect to various peptide substrates. The cosubstrates needed by the enzyme in vivomay be provided by either the virus or more likely by its host. On the basis of these data it seems very probable that the occurrence of 4-hydroxyproline in proteins is not restricted to certain animal and plant proteins." @default.
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