Separation of prolyl 3-hydroxylase and 4-hydroxylase activities and the 4-hydroxyproline requirement for synthesis of 3-hydroxyproline

Differences between prolyl 3-hydroxylase and prolyl 4-hydroxylase activities were found in their stimulation and inactivation by dithiothreitol and in their affinity to poly-L-proline linked to agarose. The two enzyme activities were separated by gel filtration, the results demonstrating that they are due to separate proteins. Comparison of [¹⁴C]proline-labelled protocollagen and the same protein when fully 4-hydroxylated as substrates indicated dependence of 3-hydroxyproline formation on the presence of 4-hydroxyproline. It is suggested that the main substrate sequence for 3-hydroxyproline synthesis is -Gly-Pro-4Hyp-Gly-.     

Inhibition of prolyl 4-hydroxylase by hydroxyanthraquinones.

Prolyl 4-hydroxylase (EC 1.14.11.2) is an essential enzyme in the post-translational modification of collagen. Inhibitors of this enzyme are of potential interest for the treatment of diseases involving excessive deposition of collagen. We have found that anthraquinones with at least two hydroxy groups ortho to each other are potent inhibitors of this enzyme. Kinetic studies revealed that 2,7,8-trihydroxyanthraquinone (THA) competitively inhibited the co-substrate, 2-oxoglutarate, but was non-competitive with regard to ascorbate and was tentatively considered to be uncompetitive with regard to protocollagen. The inhibition by THA was greatly enhanced in the absence of added Fe2+ and was partially reversed by the addition of concentrations of Fe2+ in excess of the optimum for the enzymic reaction. Binding studies indicated that THA is an effective chelating agent for Fe2+. Several non-quinoidal compounds bearing the catechol moiety also inhibited the enzyme. The results suggest that THA inhibited prolyl 4-hydroxylase by binding to the enzyme at the site for 2-oxoglutarate possibly involving the Fe2+ atom, rather than by complexing with Fe2+ in free solution. The inhibition of prolyl 4-hydroxylase by THA exhibited strong positive co-operativity and may involve three distinct but non-independent binding sites.     

Interaction of prolyl 4-hydroxylase with synthetic peptide substrates. A conformational model for collagen proline hydroxylation.

With the aim of understanding the structural basis for the substrate specificity of collagen prolyl 4-hydroxylase, we have studied the conformational features of synthetic oligopeptide substrates and their interaction with the enzyme purified from chicken embryo. Circular dichroism and infrared spectral data, taken in conjunction with relevant crystal structure data, indicated an equilibrium mixture of the polyproline-II (PP-II) helix, the beta-turn, and the random coil conformations in aqueous and trifluoroethanol solutions of the "collagen-related" peptides: t-Boc-Pro-Pro-Gly-Pro-OH, t-Boc-Pro-Pro-Gly-Pro-NHCH3, t-Pro-Pro-Gly-Pro-Pro-OH, t-Boc-Pro-Pro-Ala-Pro-OH, and t-Boc-Pro-Pro-Gln-Pro-OCH3, where t-Boc is tert-butoxycarbonyl. In another set of peptides related to elastin, t-Boc-Val-Pro-Gly-Val-OH and t-Boc-Gly-Val-Pro-Gly-Val-OH, the data indicated the beta-structure, rather than the PP-II helix, was in equilibrium with the beta-turn. Kinetic parameters for the enzymatic hydroxylation of the peptides showed that as a group, the first (proline-rich) set of peptides has higher Km values and lower Vmax and Kcat/Km values than the valine-rich peptides. Data on the inhibition of hydroxylation of the standard assay substrate (Pro-Pro-Gly)10 by the oligopeptides pointed to common binding sites for the peptides. Hydroxyproline-containing peptides had no effect on the hydroxylation of the standard substrate, showing the absence of product inhibition. Based on these and earlier data, we propose that in collagen and related peptides, a supersecondary structure consisting of the PP-II helix followed by the beta-turn is the minimal structural requirement for proline hydroxylation. The PP-II structure would aid effective interaction at the substrate binding subsites, while the beta-turn would be essential at the catalytic site of the enzyme. In elastin and related peptides, the beta-strand structure may be interchangeable with the PP-II structure. This conformational model for proline hydroxylation resolves the discrepancies in earlier proposals on the substrate specificity of prolyl 4-hydroxylase. It is also consistent with the available information on the active site geometry of the enzyme.     

Syncatalytic inactivation of prolyl 4-hydroxylase by anthracyclines.

The anthracyclines doxorubicin and daunorubicin were found to act as irreversible inhibitors of prolyl 4-hydroxylase. The reaction rate for enzyme from both chick and human origin was first order, the concentration of inhibitor giving 50% inhibition being 60 microM for both compounds after 1 h. The effect was dependent on the presence of iron ions in the reaction mixture. Inactivation could be prevented by addition of high concentrations of ascorbate, but not 2-oxoglutarate, before the inactivation period. The same results were obtained with competitive analogues of these cosubstrates. Lysyl hydroxylase from chick embryos was also susceptible to inactivation. Its activity was decreased by 50% after incubation for 1 h with a 150 microM concentration of the inhibitors. When chick-embryo prolyl 4-hydroxylase was incubated with [14-14C]doxorubicin, both enzyme subunits were radioactively labelled, about 70% of the total radioactivity being found in the alpha-subunit. Since the anthracyclines are known to undergo a redox reaction generating semiquinone radicals with Fe3+ only, the results suggest that the enzyme-bound iron ion is oxidized to a tervalent intermediate in uncoupled reaction cycles. The data also suggest that both enzyme subunits contribute to the catalytic site of prolyl 4-hydroxylase.     

Subcellular localization of procollagen I and prolyl 4-hydroxylase in corneal endothelial cells

To investigate the molecular mechanism of intracellular degradation of type I collagen in normal corneal endothelial cells (CEC), we studied the role of prolyl 4-hydroxylase (P4-H) and protein disulfide-isomerase (PDI; the beta subunit of P4-H) during procollagen I biosynthesis. When the subcellular localization of P4-H and PDI was determined, P4-H demonstrated a characteristic diffuse endoplasmic reticulum (ER) pattern, whereas PDI showed a slightly more restricted distribution within the ER. When colocalization of procollagen I with the enzymes was examined, procollagen I and PDI showed a large degree of colocalization. P4-H and procollagen I were predominantly colocalized at the perinuclear site. When colocalization of type IV collagen with PDI and P4-H was examined, type IV collagen was largely colocalized with PDI, which showed a wider distribution than type IV collagen. Type IV collagen is similarly colocalized with P4-H, except in some perinuclear sites. The colocalization profiles of procollagen I with both PDI and P4-H were not altered in cells treated with alpha,alpha'-dipyridyl compared to those of the untreated cells. The underhydroxylated type IV collagen demonstrated a colocalization profile with PDI similar to that observed with procollagen I, while the underhydroxylated type IV collagen was predominantly colocalized with P4-H at the perinuclear sites. Immunoblot analysis showed no real differences in the amounts of the beta subunit/PDI and the catalytic alpha subunit of P4-H in CEC compared to those of corneal stromal fibroblasts (CSF). When protein-protein association was determined, procollagen I was associated with PDI much more in CEC than it was in CSF, whereas type IV collagen showed no differential association specificity to PDI in both cells. Limited proteolysis of the newly synthesized intracellular procollagen I with pepsin showed that procollagen I in CEC was degraded by pepsin, whereas CSF contained type I collagen composed of alpha1(I) and alpha2(I). These findings suggest that procollagen I synthesized in CEC is not in triple helical conformation and that the improperly folded procollagen I may be preferentially associated with PDI before targeting to the intracellular degradation.     

Partial purification and characterization of chick-embryo prolyl 3-hydroxylase.

Prolyl 3-hydroxylase was purified up to about 5000-fold from an (NH4)2SO4 fraction of chick-embryo extract by a procedure consisting of affinity chromatography on denatured collagen linked to agarose, elution with ethylene glycol and gel filtration. The molecular weight of the purified enzyme is about 160000 by gel filtration The enzyme is probably a glycoprotein, since (a) its activity is inhibited by concanavalin A, and (b) the enzyme is bound to columns of this lectin coupled to agarose and can be eluted with a buffer containing methyl alpha-D-mannoside. The Km values for Fe2+, 2-oxoglutarate, O2 and ascorbate in the prolyl 3-hydroxylase reaction were found to be very similar to those previously reported for these co-substrates in the prolyl 4-hydroxylase and lysyl hydroxylase reactions.     

Microbial proline 4-hydroxylase screening and gene cloning.

Microbial proline 4-hydroxylases, which hydroxylate free L-proline to trans-4-hydroxy-L-proline, were screened in order to establish an industrial system for biotransformation of L-proline to trans-4-hydroxy-L-proline. Enzyme activities were detected in eight strains, including strains of Dactylosporangium spp. and Amycolatopsis spp. The Dactylosporangium sp. strain RH1 enzyme was partially purified 3,300-fold and was estimated to be a monomer polypeptide with an apparent molecular mass of 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Degenerate primers based on the N-terminal amino acid sequence of the 31-kDa polypeptide were synthesized in order to amplify the corresponding 71-bp DNA fragment. A 5.5-kbp DNA fragment was isolated by using the 71-bp fragment labeled with digoxigenin as a probe for a genomic library of Dactylosporangium sp. strain RH1 constructed in Escherichia coli. One of the open reading frames found in the cloned DNA, which encoded a 272-amino-acid polypeptide (molecular mass, 29, 715 daltons), was thought to be a proline 4-hydroxylase gene. The gene was expressed in E. coli as a fused protein with the N-terminal 34 amino acids of the beta-galactosidase alpha-fragment. The E. coli recombinant exhibited proline 4-hydroxylase activity that was 13. 6-fold higher than the activity in the original strain, Dactylosporangium sp. strain RH1. No homology was detected with other 2-oxoglutarate-dependent dioxygenases when databases were searched; however, the histidine motif conserved in 2-oxoglutarate-dependent dioxygenases was found in the gene.     

A rapid assay for prolyl 3-hydroxylase activity.

An assay is reported for prolyl 3-hydroxylase activity. The method is based on the release of tritiated water (THO) during 3-hydroxylation of a 2,3-T-l-proline-labeled (T = tritium) polypeptide substrate in which all prolyl residues recognized by prolyl 4-hydroxylase have been converted to 4-hydroxyprolyl residues. The formation of THO was essentially linear with enzyme concentration and time, and the K_m for the polypeptide substrate was about 3.4 × 10^?8m. A linear correlation was found between THO release and the synthesis of 3-hydroxyproline, the latter being analyzed by amino acid analyzer. The assay is simple, rapid, sensitive, and reproducible, and it is specific even in tissue samples containing a large excess of prolyl 4-hydroxylase activity.     

Direct and continuous assay for prolyl 4-hydroxylase

Prolyl 4-hydroxylase (P4H) is a nonheme iron dioxygenase that catalyzes the posttranslational hydroxylation of (2S)-proline (Pro) residues in protocollagen strands. The resulting (2S,4R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. P4H uses α-ketoglutarate and O₂ as cosubstrates, and forms succinate and CO₂ as well as Hyp. Described herein is the first assay for P4H that continuously and directly detects turnover of the proline-containing substrate. This assay is based on (2S,4S)-4-fluoroproline (flp), a proline analogue that is transformed into (2S)-4-ketoproline (Kep) and inorganic fluoride by P4H. The fluoride ion, and thus turnover by P4H, is detected by a fluoride ion-selective electrode. Using this assay, steady-state kinetic parameters for the human P4H-catalyzed turnover of a flp-containing peptide were determined and found to be comparable to those obtained with a discontinuous HPLC-based assay. In addition, this assay can be used to characterize P4H variants, as demonstrated by a comparison of catalysis by D414A P4H and the wild-type enzyme. Finally, the use of the assay to identify small-molecule inhibitors of P4H was verified by an analysis of catalysis in the presence of 2,4-pyridine dicarboxylate, an analogue of α-ketoglutarate. Thus, the assay described herein could facilitate biochemical analyses of this essential enzyme.     

Production of human prolyl 4-hydroxylase in Escherichia coli

Prolyl 4-hydroxylase (P4H) catalyzes the post-translational hydroxylation of proline residues in collagen strands. The enzyme is an alpha2beta2 tetramer in which the alpha subunits contain the catalytic active sites and the beta subunits (protein disulfide isomerase) maintain the alpha subunits in a soluble and active conformation. Heterologous production of the native alpha2beta2 tetramer is challenging and had not been reported previously in a prokaryotic system. Here, we describe the production of active human P4H tetramer in Escherichia coli from a single bicistronic vector. P4H production requires the relatively oxidizing cytosol of Origami B(DE3) cells. Induction of the wild-type alpha(I) cDNA in these cells leads to the production of a truncated alpha subunit (residues 235-534), which assembles with the beta subunit. This truncated P4H is an active enzyme, but has a high Km value for long substrates. Replacing the Met235 codon with one for leucine removes an alternative start codon and enables production of full-length alpha subunit and assembly of the native alpha2beta2 tetramer in E. coli cells to yield 2 mg of purified P4H per liter of culture (0.2 mg/g of cell paste). We also report a direct, automated assay of proline hydroxylation using high-performance liquid chromatography. We anticipate that these advances will facilitate structure-function analyses of P4H.     

Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [alpha(I)]2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [alpha(II)]2beta2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids. Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module. Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase. Keywords: collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase     

High-level production of human collagen prolyl 4-hydroxylase in Escherichia coli.

The collagen prolyl 4-hydroxylases (C-P4Hs), enzymes residing within the lumen of the endoplasmic reticulum, play a central role in the synthesis of all collagens. The vertebrate enzymes are alpha(2)beta(2) tetramers in which the two catalytic sites are located in the alpha subunits, and protein disulfide isomerase serves as the beta subunit. All attempts to assemble an active C-P4H tetramer from its subunits in in vitro cell-free systems have been unsuccessful, but assembly of a recombinant enzyme has been reported in several cell types by coexpression of the two types of subunit. An active type I C-P4H tetramer was obtained here by periplasmic expression in Escherichia coli strains BL21 and RB791. Further optimization for production by stepwise regulated coexpression of its subunits in the cytoplasm of a thioredoxin reductase and glutathione reductase mutant E. coli strain resulted in large amounts of human type I C-P4H tetramer. The specific activity of the C-P4H tetramer purified from the cytoplasmic expression was within the range of values reported for human type I C-P4H isolated as a nonrecombinant enzyme or produced in the endoplasmic reticulum of insect cells, but the expression level, about 25 mg/l in a fermenter, is about 5-10 times that obtained in insect cells. The enzyme expressed in E. coli differed from those present in vivo and those produced in other hosts in that it lacked the N glycosylation of its alpha subunits, which may be advantageous in crystallization experiments.     

Prolyl 3-hydroxylase and 4-hydroxylase activities in certain rat and chick-embryo tissues and age-related changes in their activities in the rat.

Prolyl 3-hydroxylase activity, expressed per unit of extract protein, was much higher in rat kidney cortex than in the lung, liver or skin. A marked decrease in activity was found in the kidney cortex, liver and skin beyond 10 days of age. The ratio of prolyl 3-hydroxylase to 4-hydroxylase activity in the kidney cortex was 13--17 times that in the skin, that in the liver 6--8 times, and that in the lung about twice the value for the skin, there being no changes in this ratio with age. In 16-day chick embryos the highest ratios of prolyl 3-hydroxylase to 4-hydroxylase activity were found in the liver, heart, lens, aorta and kidney, and the lowest ratios in tendon, cartilage, cartilaginous and membranous bone and skin. The results suggest that the differences in the extent of prolyl 3-hydroxylation between various collagens can in part be explained by differences in the amount of prolyl e-hydroxylase activity among different cells.     

Conformational preferences of substrates for human prolyl 4-hydroxylase

Prolyl 4-hydroxylase (P4H) catalyzes the posttranslational hydroxylation of (2 S)-proline (Pro) residues in procollagen strands. The resulting (2 S,4 R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. Even though its product (Hyp) differs from its substrate (Pro) by only a single oxygen atom, no product inhibition has been observed for P4H. Here, we examine the basis for the binding and turnover of substrates by human P4H. Synthetic peptides containing (2 S,4 R)-4-fluoroproline (Flp), (2 S,4 S)-4-fluoroproline (flp), (2 S)-4-ketoproline (Kep), (2 S)-4-thiaproline (Thp), and 3,5-methanoproline (Mtp) were evaluated as substrates for P4H. Peptides containing Pro, flp, and Thp were found to be excellent substrates for P4H, forming Hyp, Kep, and (2 S,4 R)-thiaoxoproline, respectively. Thus, P4H is tolerant to some substitutions on C-4 of the pyrrolidine ring. In contrast, peptides containing Flp, Kep, or Mtp did not even bind to the active site of P4H. Each proline analogue that does bind to P4H is also a substrate, indicating that discrimination occurs at the level of binding rather than turnover. As the iron(IV)-oxo species that forms in the active site of P4H is highly reactive, P4H has an imperative for forming a snug complex with its substrate and appears to do so. Most notably, those proline analogues with a greater preference for a C (gamma)- endo pucker and cis peptide bond were the ones recognized by P4H. As Hyp has a strong preference for C (gamma)- exo pucker and trans peptide bond, P4H appears to discriminate against the conformation of proline residues in a manner that diminishes product inhibition during collagen biosynthesis.     

Effect of prevention of procollagen triple-helix formation on proline 3-hydroxylation in freshly isolated chick-embryo tendon cells.

Inhibition of procollagen triple-helix formation by the addition of cis-hydroxyproline or azetidine-2-carboxylic acid increased the synthesis of 3-hydroxy[14C]proline 1.7-1.8-fold in pulse-chase experiments with freshly isolated chick-embryo tendon cells. The amount of 3-hydroxy[14C]proline, expressed as a percentage of the total 14C radioactivity in hydroxyproline, reached 8.4%. Control experiments indicated that the two analogues had no effect on the prolyl 3-hydroxylase activity of these cells. The data suggest that the time available before triple-helix formation in part regulates the extent of the 3-hydroxylation of proline in the biosynthesis of collagen in intact cells.     

Enzymatic production of trans-4-hydroxy-l-proline by proline 4-hydroxylase

Trans-4-hydroxy-l -proline (Hyp) is a useful chiral building block for production of many nutritional supplements and pharmaceuticals. However, it is still challenging for industrial production of Hyp due to heavy environmental pollution and low production efficiency. To establish a green and efficient process for Hyp production, the proline 4-hydroxylase (DsP4H) from Dactylosporangium sp. RH1 was overexpressed and functionally characterized in Escherichia coli BL21(DE3). The recombinant DsP4H with l -proline as a substrate exhibited K_m, k_cat and k_cat/K_m values up to 0.80 mM, 0.52 s⁻¹ and 0.65 s⁻¹·mM⁻¹ respectively. Furthermore, DsP4H showed the highest activity at 35°C and pH 6.5 towards l -proline. The highest enzyme activity of 175.6 U mg⁻¹ was achieved by optimizing culture parameters. Under the optimal transformation conditions in a 5-l fermenter, Hyp titre, conversion rate and productivity were up to 99.9 g l⁻¹, 99.9% and 2.77 g l⁻¹ h⁻¹ respectively. This strategy described here provides an efficient method for production of Hyp and thus has a great potential in industrial application.