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PubMed2000

Construction of a novel hydroxyproline-producing recombinant Escherichia coli by introducing a proline 4-hydroxylase gene.

An Escherichia coli recombinant strain producing trans-4-hydroxy-L-proline (Hyp) was constructed by introducing a proline 4-hydroxylase gene into an L-proline-producing E. coli. Plasmid pPF1, which contains a gene encoding feedback resistant gamma-glutamyl kinase (proB74), was constructed and introduced into E. coli W1485 putA. The recombinant E. coli W1485 putA/pPF1 strain produced L-proline (1.2 g/l). The proline production by W1485 putA/pPF1 was converted to Hyp production by introducing pWFH1 which contains a proline 4-hydroxylase gene. E. coli W1485 putA which harbors pWFP1 carrying the proline 4-hydroxylase gene, proB74, and proA produced 25 g/l of Hyp in 96 h. A novel biosynthetic pathway of Hyp, which has not previously been produced in E. coli, was constructed in E. coli.

Journal Article↗

PubMed2002

Effect of proline and arginine metabolism on freezing stress of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the PUT1-encoded proline oxidase and the PUT2-encoded delta1-pyrroline-5-carboxylate dehydrogenase are required to convert proline to glutamate. We recently showed that a put1 disruptant accumulated higher levels of proline intracellularly and conferred higher resistance to freezing stress. Here, we determined the effect of put2 disruption on yeast cell viability under freezing stress. When grown on arginine as the sole nitrogen source, the put2 disruptant showed a significant decrease in cell viability after freezing despite the high proline and arginine contents. This result suggests that delta1-pyrroline-5-carboxylate or glutamate-gamma-semialdehyde, a proline catabolism intermediate, is toxic to yeast cells under freezing stress. In contrast, the survival rate of the wild-type and the put1-disruptant strains was found to increase after freezing in proportion to their arginine contents. This indicates that arginine has a cryoprotective function in yeast. Furthermore, the yeast cells accumulated proline as well as arginine in the vacuole, suggesting that there is a system for the transport of excess proline to the vacuole and that this vacuolar accumulation may be important in the freezing resistance of yeast cells.

Journal Article↗

PubMed1976

Contribution of Arginine to Proline Accumulation in Water-stressed Barley Leaves.

Barley (Hordeum vulgare cv Prior) leaves converted l-U-(14)C-arginine to labeled proline. Accumulation of radioactivity in proline was greater in wilted leaves, but only after 9 hours of incubation. As the increase in free proline was detectable after only 3 to 6 hours, it is likely that the observed stimulation of proline labeling represents a result rather than a cause of proline accumulation. Furthermore, the loss of total arginine during water stress was only 10 to 15% of the increase in proline. We conclude that arginine probably contributes less than 1% of the carbon in the expanding proline pool of wilted barley leaves.

Journal Article↗

PubMed1979

Proline Accumulation in Water-stressed Barley Leaves in Relation to Translocation and the Nitrogen Budget.

Mobilization of N from leaves of barley (Hordeum vulgare L.) during water stress, and the role of proline as a mobilized species, were examined in plants at the three-leaf stage. The plants responded to water stress by withdrawing about 25% of the total reduced N from the leaf blades via phloem translocation. Most of this N loss was during the first 2 days while translocation of (14)C-photosynthate out of the stressed blade still remained active. Free proline accumulation in the blade was initially slow, and became more rapid during the 2nd day of stress. Although a major free amino acid, proline accounted for only about 5% of the total N (soluble + insoluble) retained in severely stressed blades. When the translocation pathway in water-stressed leaves was interrupted just below the blade by a heat girdle, a cold jacket, or by blade excision, N loss from the blade was prevented and proline began to accumulate rapidly on 1st day of stress. Little free proline accumulated in the blades until after the ability to translocate was lost. Proline was, however, probably not a major species of N translocated during stress, because proline N accumulation in heat-girdled stressed leaves was five times slower than the rate of total N export from intact blades.

Journal Article↗

PubMed1983

Selective inhibition of proline hydroxylation by 3,4-dehydroproline.

The effect of proline analogs on peptidyl proline hydroxylation has been studied in vivo using aerated root slices of Daucus carota. One analog, 3,4-dehydroproline, acted at micromolar concentrations to rapidly and selectively inhibit peptidyl proline hydroxylation. A structurally altered hydroxyproline-rich cell wall glycoprotein was synthesized and secreted by dehydroproline-treated tissue. The capacity to hydroxylate proline recovered slowly following a short pulse treatment with the analog, with a halftime for recovery of about 24 hours. Recovery was not altered by supplying exogenous proline. Dehydroproline had little effect on the induction of nitrate reductase by nitrate, nor on wound-induced increases in amino acid uptake and protein synthesis. In contrast, other proline analogs inhibit proline hydroxylation only at millimolar concentrations. It is hypothesized that dehydroproline acts as an enzyme-activated suicide inhibitor of prolyl hydroxylase. This analog should become a useful tool for elucidating the functional significance of hydroxyproline-rich glycoproteins.

Journal Article↗

PubMed1984

Steady state proline levels in salt-shocked barley leaves.

Excised barley (Hordeum vulgare var Larker) leaves were treated with salt solutions or wilted. After the treatment period, the leaves were allowed to recover in a 50 millimolar sucrose and 1 millimolar glutamate solution, and proline, Na(+), and K(+) were measured at intervals. Na(+) and K(+) concentrations stayed at a constant high level after the salt treatments, and proline increased to a steady state concentration in response. The relationship between the maximum rate of proline accumulation and the Na(+) concentration reached in each experiment was linear. The final steady state proline concentration reached was also directly proportional to the Na(+) concentration. For a given Na(+) concentration in the leaves, the steady state proline level was greater when 410 millimolar NaCl was added to the leaves than when 205 millimolar NaCl was added. These results are consistent with proline acting as a compatible cytoplasmic solute, balancing an accumulation of salts outside of the cytoplasm.In contrast to the proline levels in salt-shocked leaves, the concentrations in wilted leaves decreased to near control levels within 24 hours of relief of stress.

Journal Article↗

PubMed1987

Abscisic Acid accumulation is not required for proline accumulation in wilted leaves.

Leaves from dark-grown barley (Hordeum vulgare L. var Larker) seedlings grown in the presence and absence of fluridone were used to determine whether or not abscisic acid (ABA) accumulation was necessary for proline to accumulate in wilted tissue. Wilted tissue (polyethylene glycol-treated) leaves from fluridone-grown seedlings did not accumulate ABA but did accumulate proline at a rate that was not different from the non-fluridone-treated leaves. Thus ABA accumulation is not required for wilting-induced proline accumulation in barley leaves. Proline accumulation in wilted leaves from the wilty tomato (Lycopersicon esculentum) mutant, flacca, was compared to that in the wild type, Rheinlands Ruhm. Proline accumulated in wilted leaves from flacca. The rate of accumulation was faster in flacca compared to the rate in the wild type because the wilty mutant wilted faster. ABA accumulated in wilted leaves from the wild type but not in the wilty mutant. This result is a further confirmation that ABA accumulation is not required for wilting-induced proline accumulation. These results are significant in that proline accumulation in barley leaves can be induced independently by any one of three treatments: wilting, ABA, or salt.

Journal Article↗

PubMed1985

Poly(L-proline)-binding proteins from chick embryos are a profilin and a profilactin.

Two poly(L-proline)-binding proteins (PBP-1 and PBP-2) were purified from chick embryos by using a poly(L-proline)-agarose column. PBP-1 was composed of two different polypeptides (molecular masses of 42 kDa and 15 kDa). The molar ratio of the two proteins in the complex was 1:1. The other poly(L-proline)-binding protein, PBP-2, was the 15-kDa protein itself. The 42-kDa protein was confirmed to be an actin from the amino acid composition, by immunochemical evidence and by its ability to self-polymerize. In addition, the 42 + 15-kDa protein complex (PBP-1) inhibited DNase I, just as a monomeric actin did. The amino acid composition of the 15-kDa protein was similar to that of mammalian profilin and it inhibited the salt-induced polymerization of rabbit skeletal muscle actin. Therefore, we conclude that the two poly(L-proline)-binding proteins from the chick embryo are a profilactin and a profilin in chick embryo. The ability of profilactin to bind poly(L-proline) must be due to profilin itself, because the profilin has a greater affinity for poly(L-proline) than does profilactin. Additionally, both the monomeric and filamentous actin from rabbit skeletal muscle have no affinity for poly(L-proline).

Actins↗

PubMed1979

Biosynthesis of trans-4-hydroxy-L-proline by Streptomyces griseoviridus.

Radioisotopic experiments have revealed that free trans-4-hydroxy-L-proline is an intermediate synthesized from L-proline during formation of the peptide-bound cis-4-hydroxy-D-proline residue in the antibiotic, etamycin. This conclusion was based on the fact that 1) both radiolabeled L-proline and trans-4-hydroxy-L-proline are precursors of the bound D-imino acid as noted previously by Hook and Vining ((1973) J. Chem. Soc. Chem. Commun. 185-186; (1973) Can. J. Biochem. 51, 1630-1637), 2) the unlabeled trans isomer specifically inhibited the incorporation of radiolabel from proline into the antibiotic, 3) the 14C-hydroxyimino-acid was isolated from the intracellular pool and medium following incubations with L-[14C]proline during antibiotic biosynthesis and when etamycin synthesis was blocked by D-leucine. By means of chromatographic and enzymatic analyses, it was established that the free imino acid possesses the trans-L configuration.

Amino Acids↗

PubMed1980

The subcellular and organ distribution and natural form of histidyl-proline diketopiperazine in rat brain determined by a specific radioimmunoassay.

Histidyl-proline diketopiperazine is produced in brain as a product of the metabolism of thyrotropin-releasing hormone. A number of the previously observed central nervous system and pituitary activities resulting from an exposure to thyrotropin-releasing hormone appear to involve the conversion of the releasing factor to the cyclic dipeptide. In the present study, the development of a rabbit antiserum that is highly specific for histidyl-proline diketopiperazine is described; the antiserum has essentially no capability to bind thyrotropin-releasing hormone or a number of other related peptides. The antibody can also distinguish between the natural form of the cyclic dipeptide and a diastereomer containing D-proline. A procedure for extraction, with high yield, of histidyl-proline diketopiperazine from brain is described. With the aid of the specific antiserum it was found that the preponderance of the cyclic dipeptide in rat brain is bound to high molecular weight material, mainly in the range of Mr = 70,000; histidyl-proline diketopiperazine can be disassociated from this material by boiling in salt/methanol solution. The concentration of the dipeptide in rat brain is in the range of 275 to 565 pmol/brain, approximately 2.5 times the concentrations determined for thyrotropin-releasing hormone (113 to 210 pmol/brain). A study of the subcellular distribution of histidyl-proline diketopiperazine and thyrotropin-releasing hormone suggests that the releasing factor is concentrated in synaptosomal vesicles while the diketopiperazine is not. A determination of the regional distribution of thyrotropin-releasing hormone and histidyl-proline diketopiperazine indicated that both peptides are found in highest concentrations in pituitary and hypothalamus, but are detectable in other areas of brain as well.

Animals↗

PubMed1995

Scanning tunneling microscopy study of poly-L-proline.

Differences in its peptide bonds allow the imino acid poly-L-proline to exist in two significantly different geometric structures. Form I with cis peptide bonds is supposed to be a right-handed helix and form II with trans peptide bonds a left-handed helix. Cis/trans isomerization about the proline imide is believed to cause the denaturation of a number of proteins and may be a key step in protein folding. Using scanning tunneling microscopy (STM), we present high-resolution images of air-dried poly-L-proline. It is found that the electric conductivity of one monolayer of poly-L-proline is sufficient to allow for STM imaging without significant tip-sample interaction. Only at locations where stacking of poly-L-proline chains occurs, a direct contact of the probing tip to the molecules becomes apparent and prevents us, at present, from resolving the atomic structure of the topmost layer. Our STM images of poly-L-proline show that form II is relatively rigid and forms aggregates in most cases. Form I, which is occasionally observed, is very flexible and exhibits sharp bends as well as 180 degrees backfolding. These observations confirm theoretical predictions on the existence of two peptide bond conformations of poly-L-proline.

Isomerism↗

PubMed1982

Salivary proline-rich proteins.

Proline-rich proteins are major components of parotid and submandibular saliva in humans as well as other animals. They can be divided into acidic, basic and glycosylated proteins. The primary structure of the acidic proline-rich proteins is unique and shows that the proteins do not belong to any known family of proteins. The proline-rich proteins are apparently synthesized the acinar cells of the salivary glands and their phenotypic expression is under complex genetic control. The acidic proline-rich proteins will bind calcium with a strength which indicates that they may be important in maintaining the concentration of ionic calcium in saliva. Moreover they can inhibit formation of hydroxyapatite, whereby growth of hydroxyapatite crystals on the tooth surface in vivo may be avoided. Both of these activities as well as the binding site for hydroxyapatite are located in the N-terminal proline-poor part of the protein. Little is known about the functions of the glycosylated and basic proline-rich proteins.

Amino Acids↗

PubMed1980

A new prolyl hydroxylase acting on poly-L-proline, from suspension cultured cells of Vinca rosea.

A new prolyl hydroxylase having a novel substrate specificity was isolated from the suspension-cultured cells of Vinca rosea. This enzyme was solubilized with 0.05 M Tris-HCl buffer (pH 7.4) containing 0.1% Triton X-100, 0.3 M NaCl and 0.5 mM beta-mercaptoethanol from the membrane fractions of the cells, and was partially purified by (NH4)2SO4 fractionation and DEAE-Sephadex A-50 column chromatography. The enzyme preparation was found to require O2, Fe2+, ascorbate, alpha-ketoglutarate and poly-L-proline to attain maximum activity. The plant enzyme does not hydroxylate free proline and di-, tri- and tetra-L-proline, but hydroxylates octa-L-proline and poly-L-proline (Mr greater than 2000). Model peptides of unhydroxylated collagen, (Pro-Pro-Gly)5 and (Pro-Pro-Gly)10 are poor substrates for the plant enzyme. This means that the plant enzyme has a novel substrate specificity in regard to peptidyl substrate, and this differs from vertebrate prolyl hydroxylase, proline,2-oxoglutarate dioxygenase (prolyl-glycyl-peptide, 2-oxoglutarate: oxygen oxidoreductase, EC 1.14.11.2).

Ascorbic Acid↗

PubMed1991

Schistosoma mansoni: higher free proline levels in the livers of infected mice.

The concentration of L-hydroxyproline in the liver of ICR female mice increased rapidly during the 8th to 11th weeks of Schistosoma mansoni infection. Free L-proline concentration began to increase about the 7th week and reached its maximum at the 8th to 9th weeks of the infection, when the granulomatous response to the schistosome eggs in the liver was most prominent, as indicated by the increase in liver wet weight and its deoxyribonucleic acid concentration. A significant increment in the total activity of ornithine-delta-transaminase (EC 2.6.1.13) and the decrease in the specific activity of proline oxidase (EC 1.4.3.2) became detectable in the liver homogenate of infected mice on the 8th week. However, changes in these enzymatic activities were not parallel to that of the hepatic free L-proline content. Intraperitoneal administration of S. mansoni egg granulomas or 15,000g x 30 min supernatant fluid of their extracts into uninfected, normal mice significantly increased the hepatic free L-proline content without any appreciable effect on the enzymatic activities of proline oxidase and ornithine-delta-transaminase. These findings suggest that S. mansoni egg granulomas contain a factor(s) which may be responsible for the elevation of free L-proline content in the fibrotic liver caused by experimental schistosomiasis mansoni.

Animals↗

PubMed2004

Structures of the Escherichia coli PutA proline dehydrogenase domain in complex with competitive inhibitors.

Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the flavin-dependent oxidation of proline to Delta(1)-pyrroline-5-carboxylate. Here we present a structure-based study of the PRODH active site of the multifunctional Escherichia coli proline utilization A (PutA) protein using X-ray crystallography, enzyme kinetic measurements, and site-directed mutagenesis. Structures of the PutA PRODH domain complexed with competitive inhibitors acetate (K(i) = 30 mM), L-lactate (K(i) = 1 mM), and L-tetrahydro-2-furoic acid (L-THFA, K(i) = 0.2 mM) have been determined to high-resolution limits of 2.1-2.0 A. The discovery of acetate as a competitive inhibitor suggests that the carboxyl is the minimum functional group recognized by the active site, and the structures show how the enzyme exploits hydrogen-bonding and nonpolar interactions to optimize affinity for the substrate. The PRODH/L-THFA complex is the first structure of PRODH with a five-membered ring proline analogue bound in the active site and thus provides new insights into substrate recognition and the catalytic mechanism. The ring of L-THFA is nearly parallel to the middle ring of the FAD isoalloxazine, with the inhibitor C5 atom 3.3 A from the FAD N5. This geometry suggests direct hydride transfer as a plausible mechanism. Mutation of conserved active site residue Leu432 to Pro caused a 5-fold decrease in k(cat) and a severe loss in thermostability. These changes are consistent with the location of Leu432 in the hydrophobic core near residues that directly contact FAD. Our results suggest that the molecular basis for increased plasma proline levels in schizophrenic subjects carrying the missense mutation L441P is due to decreased stability of human PRODH2.

Bacterial Proteins↗

PubMed1996

The putA gene of Agrobacterium tumefaciens is transcriptionally activated in response to proline by an Lrp-like protein and is not autoregulated.

The Agrobacterium tumefaciens putA gene, which encodes proline dehydrogenase, is transcriptionally induced by exogenous proline. In contrast to the putA genes of enteric bacteria, the A. tumefaciens putA gene is not regulated by the PutA protein, as the putA promoter remained strongly proline inducible in strains lacking PutA. A putA null mutation increased the expression of the putA promoter under a variety of conditions. However, this mutation is predicted to increase the cytoplasmic concentration of proline, and this alone probably accounts for its effects on putA expression. The putA promoter was also strongly induced by valine, and the putA genotype did not affect expression by this gratuitous inducer. An open reading frame (ORF) encoding an Lrp-like protein was found transcribed divergently from putA. Disruption of this ORF, designated putR, abolished induction of the putA promoter by proline or valine. In addition to activating putA, PutR also repressed its own transcription, and this autorepression was only slightly affected by exogenous proline. The transcription start sites for the putA and putR genes are separated by 64 nucleotides, suggesting that PutR could regulate both promoters by binding to a single operator.

Agrobacterium tumefaciens↗

PubMed1962

Wound healing and collagen formation. III. A quantitative radioautographic study of the utilization of proline-H3 in wounds from normal and scorbutic guinea pigs.

The sequence of incorporation and utilization of tritium-labeled proline has been examined in healing wounds from normal and scorbutic guinea pigs. Linear incisions in the skin of the animals were allowed to heal for 7 days. Each animal was given proline-H(3), and the wounds were excised 30 minutes, 1 and 4 hours, 1, 3 and 7 days after proline administration. The tissues were fixed in osmium tetroxide, fixed again in neutral buffered formalin, embedded in epoxy resin, and sectioned at 1 micron thickness. The sections were coated with nuclear track emulsion, exposed, developed, and stained. The results of grain counts were quantitated as the number of counts per unit area overlying cells, fibers, etc. In both groups the proline reaches a maximum over the fibroblasts within 4 hours and subsequently disappears from the cells. Concomitantly, the proline reaches a maximum over the collagen (in normal animals) and extracellular fibrillar material (in scorbutic animals) by 4 hours, where it remains. The modified technique of radioautography used in this study allows not only resolution of approximately 1 micron, but also minimal background, decreased artifact, and a clear separation of the randomly situated elements within the wounds so that grain counting is facilitated. The results correlated with previous electron microscopic studies are consistent with the utilization of proline by the fibroblasts and its incorporation into collagen (in normal animals) and into the extracellular, fibrillar, non-collagenous material seen in scorbutic animals.

Animals↗

PubMed1997

Functional interactions between the proline-rich and repeat regions of tau enhance microtubule binding and assembly.

Tau is a neuronal microtubule-associated protein that promotes microtubule assembly, stability, and bundling in axons. Two distinct regions of tau are important for the tau-microtubule interaction, a relatively well-characterized "repeat region" in the carboxyl terminus (containing either three or four imperfect 18-amino acid repeats separated by 13- or 14-amino acid long inter-repeats) and a more centrally located, relatively poorly characterized proline-rich region. By using amino-terminal truncation analyses of tau, we have localized the microtubule binding activity of the proline-rich region to Lys215-Asn246 and identified a small sequence within this region, 215KKVAVVR221, that exerts a strong influence on microtubule binding and assembly in both three- and four-repeat tau isoforms. Site-directed mutagenesis experiments indicate that these capabilities are derived largely from Lys215/Lys216 and Arg221. In marked contrast to synthetic peptides corresponding to the repeat region, peptides corresponding to Lys215-Asn246 and Lys215-Thr222 alone possess little or no ability to promote microtubule assembly, and the peptide Lys215-Thr222 does not effectively suppress in vitro microtubule dynamics. However, combining the proline-rich region sequences (Lys215-Asn246) with their adjacent repeat region sequences within a single peptide (Lys215-Lys272) enhances microtubule assembly by 10-fold, suggesting intramolecular interactions between the proline-rich and repeat regions. Structural complexity in this region of tau also is suggested by sequential amino-terminal deletions through the proline-rich and repeat regions, which reveal an unusual pattern of loss and gain of function. Thus, these data lead to a model in which efficient microtubule binding and assembly activities by tau require intramolecular interactions between its repeat and proline-rich regions. This model, invoking structural complexity for the microtubule-bound conformation of tau, is fundamentally different from previous models of tau structure and function, which viewed tau as a simple linear array of independently acting tubulin-binding sites.

Amino Acid Sequence↗