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Microbial Biotechnology Mar 2021Trans-4-hydroxy-l-proline (Hyp) is a useful chiral building block for production of many nutritional supplements and pharmaceuticals. However, it is still challenging...
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 , k and k /K values up to 0.80 mM, 0.52 s and 0.65 s ·mM 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 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 , 99.9% and 2.77 g l h respectively. This strategy described here provides an efficient method for production of Hyp and thus has a great potential in industrial application.
Topics: Bacterial Outer Membrane Proteins; Bioreactors; Escherichia coli; Escherichia coli Proteins; Hydroxyproline; Proline; Prolyl Hydroxylases
PubMed: 32618422
DOI: 10.1111/1751-7915.13616 -
Plant Signaling & Behavior Nov 2012When exposed to stressful conditions, plants accumulate an array of metabolites, particularly amino acids. Amino acids have traditionally been considered as precursors... (Review)
Review
When exposed to stressful conditions, plants accumulate an array of metabolites, particularly amino acids. Amino acids have traditionally been considered as precursors to and constituents of proteins, and play an important role in plant metabolism and development. A large body of data suggests a positive correlation between proline accumulation and plant stress. Proline, an amino acid, plays a highly beneficial role in plants exposed to various stress conditions. Besides acting as an excellent osmolyte, proline plays three major roles during stress, i.e., as a metal chelator, an antioxidative defense molecule and a signaling molecule. Review of the literature indicates that a stressful environment results in an overproduction of proline in plants which in turn imparts stress tolerance by maintaining cell turgor or osmotic balance; stabilizing membranes thereby preventing electrolyte leakage; and bringing concentrations of reactive oxygen species (ROS) within normal ranges, thus preventing oxidative burst in plants. Reports indicate enhanced stress tolerance when proline is supplied exogenously at low concentrations. However, some reports indicate toxic effects of proline when supplied exogenously at higher concentrations. In this article, we review and discuss the effects of exogenous proline on plants exposed to various abiotic stresses. Numerous examples of successful application of exogenous proline to improve stress tolerance are presented. The roles played by exogenous proline under varying environments have been critically examined and reviewed.
Topics: Plants; Proline; Reactive Oxygen Species
PubMed: 22951402
DOI: 10.4161/psb.21949 -
EMBO Reports Oct 2022N-terminal sequences are important sites for post-translational modifications that alter protein localization, activity, and stability. Dipeptidyl peptidase 9 (DPP9) is...
N-terminal sequences are important sites for post-translational modifications that alter protein localization, activity, and stability. Dipeptidyl peptidase 9 (DPP9) is a serine aminopeptidase with the rare ability to cleave off N-terminal dipeptides with imino acid proline in the second position. Here, we identify the tumor-suppressor BRCA2 as a DPP9 substrate and show this interaction to be induced by DNA damage. We present crystallographic structures documenting intracrystalline enzymatic activity of DPP9, with the N-terminal Met1-Pro2 of a BRCA21-40 peptide captured in its active site. Intriguingly, DPP9-depleted cells are hypersensitive to genotoxic agents and are impaired in the repair of DNA double-strand breaks by homologous recombination. Mechanistically, DPP9 targets BRCA2 for degradation and promotes the formation of RAD51 foci, the downstream function of BRCA2. N-terminal truncation mutants of BRCA2 that mimic a DPP9 product phenocopy reduced BRCA2 stability and rescue RAD51 foci formation in DPP9-deficient cells. Taken together, we present DPP9 as a regulator of BRCA2 stability and propose that by fine-tuning the cellular concentrations of BRCA2, DPP9 alters the BRCA2 interactome, providing a possible explanation for DPP9's role in cancer.
Topics: Aminopeptidases; DNA; DNA Damage; DNA Repair; Dipeptides; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Proline; Rad51 Recombinase; Serine
PubMed: 35912982
DOI: 10.15252/embr.202154136 -
Chemical & Pharmaceutical Bulletin 2017We describe herein a manganese(IV) oxide-mediated oxidation of N-p-methoxyphenyl (PMP)-protected glycine derivatives for the synthesis of α-imino carboxylic acid...
We describe herein a manganese(IV) oxide-mediated oxidation of N-p-methoxyphenyl (PMP)-protected glycine derivatives for the synthesis of α-imino carboxylic acid derivatives. Using this methodology, utilization of unstable glyoxic acid derivatives was avoided. Furthermore, using this methodology we synthesized novel α-imino carboxylic acid derivatives such as α-imino phenyl ester, perfluoroalkyl etsers, imides, and thioester. The asymmetric Mannich reaction of those novel imine derivatives with 1,3-dicarbonyl compound is also described, and the novel α-imino imide gave improved chemical yield and stereoselectivity compared with those obtained by the use of the conventional α-imino ester-type substrate.
Topics: Amino Acids; Carbon-13 Magnetic Resonance Spectroscopy; Carboxylic Acids; Imino Acids; Proton Magnetic Resonance Spectroscopy; Spectrometry, Mass, Electrospray Ionization
PubMed: 28566650
DOI: 10.1248/cpb.c17-00158 -
The Journal of Biological Chemistry Aug 2020l-Lysine oxidase/monooxygenase (l-LOX/MOG) from sp. AIU 813 catalyzes the mixed bioconversion of l-amino acids, particularly l-lysine, yielding an amide and carbon...
l-Lysine oxidase/monooxygenase (l-LOX/MOG) from sp. AIU 813 catalyzes the mixed bioconversion of l-amino acids, particularly l-lysine, yielding an amide and carbon dioxide by an oxidative decarboxylation ( apparent monooxygenation), as well as oxidative deamination (hydrolysis of oxidized product), resulting in α-keto acid, hydrogen peroxide (HO), and ammonia. Here, using high-resolution MS and monitoring transient reaction kinetics with stopped-flow spectrophotometry, we identified the products from the reactions of l-lysine and l-ornithine, indicating that besides decarboxylating imino acids ( 5-aminopentanamide from l-lysine), l-LOX/MOG also decarboxylates keto acids (5-aminopentanoic acid from l-lysine and 4-aminobutanoic acid from l-ornithine). The reaction of reduced enzyme and oxygen generated an imino acid and HO, with no detectable C4a-hydroperoxyflavin. Single-turnover reactions in which l-LOX/MOG was first reduced by l-lysine to form imino acid before mixing with various compounds revealed that under anaerobic conditions, only hydrolysis products are present. Similar results were obtained upon HO addition after enzyme denaturation. HO addition to active l-LOX/MOG resulted in formation of more 5-aminopentanoic acid, but not 5-aminopentamide, suggesting that HO generated from l-LOX/MOG can result in decarboxylation of the imino acid, yielding an amide product, and extra HO resulted in decarboxylation only of keto acids. Molecular dynamics simulations and detection of charge transfer species suggested that interactions between the substrate and its binding site on l-LOX/MOG are important for imino acid decarboxylation. Structural analysis indicated that the flavoenzyme oxidases catalyzing decarboxylation of an imino acid all share a common plug loop configuration that may facilitate this decarboxylation.
Topics: Amino Acid Oxidoreductases; Catalysis; Hydrogen Peroxide; Hydrolysis; Mixed Function Oxygenases; Pseudomonas; Substrate Specificity
PubMed: 32527725
DOI: 10.1074/jbc.RA120.014055 -
British Journal of Clinical Pharmacology Jul 1985
Topics: Biological Availability; Captopril; Humans; Kinetics; Proline
PubMed: 3896277
DOI: 10.1111/j.1365-2125.1985.tb02805.x -
European Journal of Biochemistry Dec 1991Aminoethylcysteine, lanthionine, cystathionine and cystine are mono-deaminated either by L-amino-acid oxidase or by a transaminase exhibiting the properties described... (Review)
Review
Aminoethylcysteine, lanthionine, cystathionine and cystine are mono-deaminated either by L-amino-acid oxidase or by a transaminase exhibiting the properties described for glutamine transaminase. The deaminated products cyclize producing the respective ketimines. Authentic samples of each ketimine were prepared by reacting the appropriate aminothiol compound with bromopyruvate, except cystine ketimine which required the interaction of thiopyruvate with cystine sulfoxide. Reduction of the first three mentioned ketimines with NaBH4 yields the respective derivatives with the saturated rings of thiomorpholine and hexahydrothiazepine. The same reduction is carried out enzymically by a reductase extracted from mammalian tissues. Properties of the members of this family of compounds are described. Gas chromatography followed by mass spectrometry permits the identification of most of these products. HPLC is very useful for the determination of the ketimines by taking advantage of specific absorbance at 380 nm obtained by prior derivatization with phenylisothiocyanate. Adaptation of these and other analytical procedures to biological samples disclosed the presence of most of these compounds in bovine brain and in human urine. By using [35S]lanthionine ketimine as a representative member of the ketimine group, the specific, high-affinity, saturable and reversible binding to bovine brain membranes has been demonstrated. The binding is removed by aminoethylcysteine ketimine and by cystathionine ketimine indicating the occurrence in bovine brain of a common binding site for ketimines. The reduced ketimines are totally ineffective in competing with [35S]lanthionine ketimine. Alltogether these findings are highly indicative for the existence in mammals of a novel class of endogenous sulfur-containing cyclic products provided with a possible neurochemical function to be investigated further.
Topics: Amino Acids, Sulfur; Animals; Brain Chemistry; Humans; Imino Acids; Mammals
PubMed: 1761027
DOI: 10.1111/j.1432-1033.1991.tb16365.x -
MSphere Apr 2022An intact gut microbiota confers colonization resistance against Clostridioides difficile through a variety of mechanisms, likely including competition for nutrients....
An intact gut microbiota confers colonization resistance against Clostridioides difficile through a variety of mechanisms, likely including competition for nutrients. Recently, proline was identified as an important environmental amino acid that C. difficile uses to support growth and cause significant disease. A posttranslationally modified form, trans-4-hydroxyproline, is highly abundant in collagen, which is degraded by host proteases in response to C. difficile toxin activity. The ability to dehydrate trans-4-hydroxyproline via the HypD glycyl radical enzyme is widespread among gut microbiota, including C. difficile and members of the commensal , suggesting that this amino acid is an important nutrient in the host environment. Therefore, we constructed a C. difficile Δ mutant and found that it was modestly impaired in fitness in a mouse model of infection, and was associated with an altered microbiota when compared to mice challenged with the wild-type strain. Changes in the microbiota between the two groups were largely driven by members of the family and the genus. We found that C. difficile and type strains of three commensal had significant alterations to their metabolic gene expression in the presence of trans-4-hydroxyproline . The proline reductase () genes were elevated in C. difficile, consistent with the hypothesis that trans-4-hydroxyproline is used by C. difficile to supply proline for energy metabolism. Similar transcripts were also elevated in some commensal tested, although each strain responded differently. This suggests that the uptake and utilization of other nutrients by the commensal may be affected by trans-4-hydroxyproline metabolism, highlighting how a common nutrient may be a signal to each organism to adapt to a unique niche. Further elucidation of the differences between them in the presence of hydroxyproline and other key nutrients will be important in determining their role in nutrient competition against C. difficile. Proline is an essential environmental amino acid that C. difficile uses to support growth and cause significant disease. A posttranslationally modified form, hydroxyproline, is highly abundant in collagen, which is degraded by host proteases in response to C. difficile toxin activity. The ability to dehydrate hydroxyproline via the HypD glycyl radical enzyme is widespread among gut microbiota, including C. difficile and members of the commensal , suggesting that this amino acid is an important nutrient in the host environment. We found that C. difficile and three commensal strains had significant, but different, alterations to their metabolic gene expression in the presence of hydroxyproline . This suggests that the uptake and utilization of other nutrients by the commensal may be affected by hydroxyproline metabolism, highlighting how a common nutrient may be a signal to each organism to adapt to a unique niche. Further elucidation of the differences between them in the presence of hydroxyproline and other key nutrients will be important to determining their role in nutrient competition against C. difficile.
Topics: Animals; Clostridioides; Clostridioides difficile; Clostridium; Clostridium Infections; Hydroxyproline; Mice; Peptide Hydrolases; Proline
PubMed: 35350846
DOI: 10.1128/msphere.00926-21 -
Danish Medical Journal Apr 2013A redundant collateral network between the intestinal arteries is present at all times. In case of ischaemia in the gastrointestinal tract, the collateral blood supply... (Review)
Review
A redundant collateral network between the intestinal arteries is present at all times. In case of ischaemia in the gastrointestinal tract, the collateral blood supply can develop further, thus accommodating the demand for oxygen even in the presence of significant stenosis or occlusion of the intestinal arteries without clinical symptoms of intestinal ischaemia. Symptoms of ischemia develop when the genuine and collateral blood supply no longer can accommodate the need for oxygen. Atherosclerosis is the most common cause of obliteration in the intestinal arteries. In chronic intestinal ischaemia (CII), the fasting splanchnic blood flow (SBF) is sufficient, but the postprandial increase in SBF is inadequate and abdominal pain will therefore develop in relation to food intake causing the patient to eat smaller meals at larger intervals with a resulting weight loss. Traditionally, the CII-diagnosis has exclusively been based upon morphology (angiography) of the intestinal arteries; however, substantial discrepancies between CII-symptoms and the presence of atherosclerosis/stenosis in the intestinal arteries have been described repeatedly in the literature impeding the diagnosis of CII. This PhD thesis explores a method to determine the total SBF and its potential use as a diagnostic tool in patients suspected to suffer from CII. The SBF can be measured using a continuous infusion of a tracer and catheterisation of a hepatic vein and an artery. By measuring the SBF before and after a standard meal it is possible to assess the ability or inability to enhance the SBF and thereby diagnosing CII. In Study I, measurement of SBF was tested against angiography in a group of patients suspected to suffer from CII due to pain and weight loss. A very good agreement between the postprandial increase in SBF and angiography was found. The method was validated against a well-established method independent of the hepatic extraction of tracer using pAH in a porcine model (study II). An excellent agreement was found between the two methods for the measurement of SBF. In the same set-up metabolism and recirculation in the intestines of the 99mTechnetium labelled tracer was rejected based on the consistency between the portal and arterial contents of tracer. Based on this study we concluded that an arterial blood sample can be used instead of a portal blood sample, making the method applicable to patients. In study III, 20 healthy volunteers and 29 patients with weight loss and abdominal pain but normal morphology of the intestinal arteries were investigated. A reference value for the meal induced SBF-increase and the relation to bodyweight was established designating that bodyweight should be taken into account when diagnosing CII based on measurement of SBF. The clinical method for measuring the SBF based on hepatic 99mTc-MBF extraction is a robust method. It allows determination of the postprandial increase in SBF providing knowledge about the circulatory physiology in intestines in patients with weight loss and abdominal pain with or without intestinal arterial stenosis. Future studies within this field could include measurement of the SBF before and after revascularisation in order to quantify the effect of revascularisation or investigate whether arterial blood sampling could be avoided or the amount of blood samples (and thus the time spend) could be reduced. The three studies were presented at eleven national and international congresses and Helle Damgaard Zacho has been awarded three prizes for the presentations.
Topics: Angiography; Aniline Compounds; Animals; Anthropometry; Chronic Disease; Fasting; Glycine; Humans; Imino Acids; Intestinal Diseases; Ischemia; Organotechnetium Compounds; Oxygen Consumption; Postprandial Period; Radiopharmaceuticals; Splanchnic Circulation; Swine; p-Aminohippuric Acid
PubMed: 23651725
DOI: No ID Found -
American Journal of Physiology. Renal... Apr 2006Most neutral l-amino acid acids are transported actively across the luminal brush-border membrane of small intestine and kidney proximal tubule epithelial cells by a...
Most neutral l-amino acid acids are transported actively across the luminal brush-border membrane of small intestine and kidney proximal tubule epithelial cells by a Na(+) cotransport system named B(0) that has been recently molecularly identified (B(0)AT1, SLC6A19). We show here that the opossum kidney-derived cell line OK also displays a Na(+)-dependent B(0)-type neutral l-amino acid transport, although with a slightly differing substrate selectivity. We tested the hypothesis that one of the two B(0)AT1-related transporters, SLC6A18 (ortholog of orphan transporter XT2) or SLC6A20 (ortholog of the recently identified mammalian imino acid transporter SIT1), mediates this transport. Anti-sense RNA to OK SIT1 (oSIT1) but not to OK XT2 (oXT2) inhibited Na(+)-dependent neutral amino acid transport induced by OK mRNA injected in Xenopus laevis oocytes. Furthermore, inhibition of oSIT1 gene expression in OK cells by transfection of siRNA and expression of shRNA selectively reduced the Na(+)-dependent uptake of neutral l-amino acids. Finally, expression of OK cell oSIT1 cRNA in X. laevis oocytes induced besides the transport of the l-imino acid l-Pro also that of neutral l-amino acids. Taken together, the data indicate that in OK cells SIT1 (SLC6A20) is not only an apical imino acid transporter but also plays a major role as Na(+)-dependent neutral l-amino acid transporter. A similar double role could be envisaged for SIT1 in mammalian kidney proximal tubule and small intestine.
Topics: Amino Acid Transport Systems, Neutral; Amino Acids; Animals; Gene Expression Regulation; Kidney Tubules, Proximal; Nerve Tissue Proteins; Oocytes; Opossums; Transfection; Xenopus laevis
PubMed: 16234310
DOI: 10.1152/ajprenal.00319.2005