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Applied and Environmental Microbiology Dec 2022The -acyl-d-amino acid amidohydrolase (-d-AAase) of Variovorax paradoxus Iso1 can enantioselectively catalyze the zinc-assisted deacetylation of -acyl-d-amino acids to...
The -acyl-d-amino acid amidohydrolase (-d-AAase) of Variovorax paradoxus Iso1 can enantioselectively catalyze the zinc-assisted deacetylation of -acyl-d-amino acids to yield consistent d-amino acids. A putative FAD-binding glycine/d-amino acid oxidase was located immediately upstream of the gene. The gene encoding this protein was cloned into Escherichia coli BL21 (DE3)pLysS and overexpressed at 25°C for 6 h with 0.5 mM isopropyl β-d-1-thiogalactopyranoside induction. After purification, the tag-free recombinant protein was obtained. The enzyme could metabolize glycine, sarcosine, and d-alanine, but not l-amino acids or bulky d-amino acids. Protein modeling further supported these results, demonstrating that glycine, sarcosine, and d-alanine could fit into the pocket of the enzyme's activation site, while l-alanine and d-threonine were out of position. Therefore, this protein was proposed as a glycine oxidase, and we designated it VpGO. Interestingly, VpGO showed low sequence similarity to other well-characterized glycine oxidases. We found that VpGO and -d-AAase were expressed on the same mRNA and could be transcriptionally induced by various acids. Western blotting and zymography showed that both proteins had similar expression patterns in response to different types of inducers. Thus, we have identified a novel glycine oxidase that is co-regulated with -d-AAase in an operon, and metabolizes -acyl-d-amino acids in the metabolically versatile V. paradoxus Iso1. The Gram-negative bacterium Variovorax paradoxus has numerous metabolic capabilities, including the association with important catabolic processes and the promotion of plant growth. We had previously identified and characterized an -acyl-d-amino-acid amidohydrolase (-d-AAase) gene from the strain of V. paradoxus Iso1. The aim of this study was to isolate and characterize (both and ) another potential gene found in the promoter region of this -d-AAase gene. The protein was identified as a glycine oxidase, and the gene existed in an operon with -d-AAase. The structural basis for its FAD-binding potential and substrate stereo-specificity were also elucidated. This study first reported a novel glycine oxidase from V. paradoxus. We believe that our study makes a significant contribution to the literature, because this enzyme has great potential for use as an industrial catalysis, as a biosensor, and in agricultural biotechnology.
Topics: Flavin-Adenine Dinucleotide; Sarcosine; Escherichia coli; Amidohydrolases; Amino Acids; Substrate Specificity; Alanine
PubMed: 36377957
DOI: 10.1128/aem.01077-22 -
Medical Hypotheses Mar 2016Formaldehyde is extremely toxic reacting with proteins to crosslinks peptide chains. Formaldehyde is a metabolic product in many enzymatic reactions and the question of...
Formaldehyde is extremely toxic reacting with proteins to crosslinks peptide chains. Formaldehyde is a metabolic product in many enzymatic reactions and the question of how these enzymes are protected from the formaldehyde that is generated has largely remained unanswered. Early experiments from our laboratory showed that two liver mitochondrial enzymes, dimethylglycine dehydrogenase (DMGDH) and sarcosine dehydrogenase (SDH) catalyze oxidative demethylation reactions (sarcosine is a common name for monomethylglycine). The enzymatic products of these enzymes were the demethylated substrates and formaldehyde, produced from the removed methyl group. Both DMGDH and SDH contain FAD and both have tightly bound tetrahydrofolate (THF), a folate coenzyme. THF binds reversibly with formaldehyde to form 5,10-methylene-THF. At that time we showed that purified DMGDH, with tightly bound THF, reacted with formaldehyde generated during the reaction to form 5,10-methylene-THF. This effectively scavenged the formaldehyde to protect the enzyme. Recently, post-translational modifications on histone tails have been shown to be responsible for epigenetic regulation of gene expression. One of these modifications is methylation of lysine residues. The first enzyme discovered to accomplish demethylation of these modified histones was histone lysine demethylase (LSD1). LSD1 specifically removes methyl groups from di- and mono-methylated lysines at position 4 of histone 3. This enzyme contained tightly bound FAD and the products of the reaction were the demethylated lysine residue and formaldehyde. The mechanism of LSD1 demethylation is analogous to the mechanism previously postulated for DMGDH, i.e. oxidation of the N-methyl bond to the methylene imine followed by hydrolysis to generate formaldehyde. This suggested that THF might also be involved in the LSD1 reaction to scavenge the formaldehyde produced. Our hypotheses are that THF is bound to native LSD1 by analogy to DMGDH and SDH and that the bound THF serves to protect the FAD class of histone demethylases from the destructive effects of formaldehyde generation by formation of 5,10-methylene-THF. We present pilot data showing that decreased folate in livers as a result of dietary folate deficiency is associated with increased levels of methylated lysine 4 of histone 3. This can be a result of decreased LSD1 activity resulting from the decreased folate available to scavenge the formaldehyde produced at the active site caused by the folate deficiency. Because LSD1 can regulate gene expression this suggests that folate may play a more important role than simply serving as a carrier of one-carbon units and be a factor in other diseases associated with low folate.
Topics: Catalytic Domain; DNA Methylation; Epigenesis, Genetic; Escherichia coli; Folic Acid; Folic Acid Deficiency; Histone Demethylases; Histones; Humans; Lysine; Mass Spectrometry; Models, Theoretical; Pilot Projects; Protein Processing, Post-Translational; Sarcosine Dehydrogenase; Tetrahydrofolates
PubMed: 26880641
DOI: 10.1016/j.mehy.2015.12.027 -
Frontiers in Bioscience (Scholar... Jan 2010Prostate cancer is the most prevalent cancer in the Western male population and the second leading cause of cancer death in men, affecting over 10 million individuals.... (Review)
Review
Prostate cancer is the most prevalent cancer in the Western male population and the second leading cause of cancer death in men, affecting over 10 million individuals. Present approaches to control the cancer mortality have focused on the detection of the cancer at early stages when it is still locally confined and may be curable. Identification of the prostate-specific antigen (PSA) has facilitated the early diagnosis of prostate cancer. However, PSA has limited specificity and sensitivity in appropriately detecting early stages of abnormal prostate growth. PSA levels fail to differentiate between indolent and aggressive cancers, do not correlate with tumor size, and cross-react with other serine proteases namely, glandular kallikreins 1 and 2. Besides cancer, its levels also increase in men with benign prostatic hyperplasia (BPH), prostatitis, and other non-malignancies. Additional prostate-specific genes and metabolites need to be identified to provide a better understanding of the molecular mechanisms of prostate physiology and pathophysiology. Novel markers for the diagnosis and development of new treatment modalities are urgently needed.
Topics: Anoctamins; Antigens, Neoplasm; Biomarkers, Tumor; Citric Acid; Endoplasmic Reticulum; Golgi Apparatus; HMGN2 Protein; Humans; Male; Membrane Proteins; Prostate-Specific Antigen; Prostatic Neoplasms; Sarcosine; Transcription Factors
PubMed: 20036976
DOI: 10.2741/s93 -
Journal of Bacteriology Jan 2016Sarcosine (N-methylglycine) is present in many environments inhabited by pseudomonads and is likely most often encountered as an intermediate in the metabolism of...
UNLABELLED
Sarcosine (N-methylglycine) is present in many environments inhabited by pseudomonads and is likely most often encountered as an intermediate in the metabolism of choline, carnitine, creatine, and glyphosate. While the enzymology of sarcosine metabolism has been relatively well studied in bacteria, the regulatory mechanisms governing catabolism have remained largely unknown. We previously determined that the sarcosine-catabolic (sox) operon of Pseudomonas aeruginosa is induced by the AraC family regulator GbdR in response to glycine betaine and dimethylglycine. However, induction of these genes was still observed in response to sarcosine in a gbdR deletion mutant, indicating that an independent sarcosine-responsive transcription factor also acted at this locus. Our goal in this study was to identify and characterize this regulator. Using a transposon-based genetic screen, we identified PA4184, or SouR (sarcosine oxidation and utilization regulator), as the sarcosine-responsive regulator of the sox operon, with tight induction specificity for sarcosine. The souR gene is required for appreciable growth on sarcosine as a carbon and nitrogen source. We also characterized the transcriptome response to sarcosine governed by SouR using microarray analyses and performed electrophoretic mobility shift assays to identify promoters directly regulated by the transcription factor. Finally, we characterized PA3630, or GfnR (glutathione-dependent formaldehyde neutralization regulator), as the regulator of the glutathione-dependent formaldehyde detoxification system in P. aeruginosa that is expressed in response to formaldehyde released during the catabolism of sarcosine. This study expands our understanding of sarcosine metabolic regulation in bacteria through the identification and characterization of the first known sarcosine-responsive transcriptional regulator.
IMPORTANCE
The Pseudomonas aeruginosa genome encodes many diverse metabolic pathways, yet the specific transcription regulators controlling their expression remain mostly unknown. Here, we used a genetic screen to identify the sarcosine-specific regulator of the sarcosine oxidase operon, which we have named SouR. SouR is the first bacterial regulator shown to respond to sarcosine, and it is required for growth on sarcosine. Sarcosine is found in its free form and is also an intermediate in the catabolic pathways of glycine betaine, carnitine, creatine, and glyphosate. The similarity of SouR to the regulators of carnitine and glycine betaine catabolism suggests evolutionary diversification within this regulatory family to allow response to structurally similar but physiologically distinct ligands.
Topics: Bacterial Proteins; Carbon; Gene Expression Regulation, Bacterial; Mutation; Nitrogen; Protein Binding; Pseudomonas aeruginosa; Sarcosine
PubMed: 26503852
DOI: 10.1128/JB.00739-15 -
The Journal of Biological Chemistry Apr 2004Sarcosine oxidase (SOX) is known as a peroxisomal enzyme in mammals and as a sarcosine-inducible enzyme in soil bacteria. Its presence in plants was unsuspected until...
Sarcosine oxidase (SOX) is known as a peroxisomal enzyme in mammals and as a sarcosine-inducible enzyme in soil bacteria. Its presence in plants was unsuspected until the Arabidopsis genome was found to encode a protein (AtSOX) with approximately 33% sequence identity to mammalian and bacterial SOXs. When overexpressed in Escherichia coli, AtSOX enhanced growth on sarcosine as sole nitrogen source, showing that it has SOX activity in vivo, and the recombinant protein catalyzed the oxidation of sarcosine to glycine, formaldehyde, and H(2) O(2) in vitro. AtSOX also attacked other N-methyl amino acids and, like mammalian SOXs, catalyzed the oxidation of l-pipecolate to Delta(1)-piperideine-6-carboxylate. Like bacterial monomeric SOXs, AtSOX was active as a monomer, contained FAD covalently bound to a cysteine residue near the C terminus, and was not stimulated by tetrahydrofolate. Although AtSOX lacks a typical peroxisome-targeting signal, in vitro assays established that it is imported into peroxisomes. Quantitation of mRNA showed that AtSOX is expressed at a low level throughout the plant and is not sarcosine-inducible. Consistent with a low level of AtSOX expression, Arabidopsis plantlets slowly metabolized supplied [(14)C]sarcosine to glycine and serine. Gas chromatography-mass spectrometry analysis revealed low levels of pipecolate but almost no sarcosine in wild type Arabidopsis and showed that pipecolate but not sarcosine accumulated 6-fold when AtSOX expression was suppressed by RNA interference. Moreover, the pipecolate catabolite alpha-aminoadipate decreased 30-fold in RNA interference plants. These data indicate that pipecolate is the endogenous substrate for SOX in plants and that plants can utilize exogenous sarcosine opportunistically, sarcosine being a common soil metabolite.
Topics: Amino Acid Sequence; Arabidopsis; Cucurbita; Cysteine; DNA, Complementary; Escherichia coli; Formaldehyde; Gas Chromatography-Mass Spectrometry; Glycine; Hydrogen Peroxide; Mass Spectrometry; Microbodies; Models, Chemical; Molecular Sequence Data; Nitrogen; Oxidoreductases Acting on CH-NH Group Donors; Oxygen; Peroxisomes; Protein Structure, Tertiary; RNA Interference; RNA, Messenger; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sarcosine; Sequence Homology, Amino Acid; Serine; Spectrometry, Mass, Electrospray Ionization; Tetrahydrofolates
PubMed: 14766747
DOI: 10.1074/jbc.M400071200 -
Frontiers in Molecular Neuroscience 2020Epileptogenesis is a common consequence of brain insults, however, the prevention or delay of the epileptogenic process remains an important unmet medical challenge....
Epileptogenesis is a common consequence of brain insults, however, the prevention or delay of the epileptogenic process remains an important unmet medical challenge. Overexpression of glycine transporter 1 (GlyT1) is proposed as a pathological hallmark in the hippocampus of patients with temporal lobe epilepsy (TLE), and we previously demonstrated in rodent epilepsy models that augmentation of glycine suppressed chronic seizures and altered acute seizure thresholds. In the present study we evaluated the effect of the GlyT1 inhibitor, sarcosine (aka N-methylglycine), on epileptogenesis and also investigated possible mechanisms. We developed a modified rapid kindling model of epileptogenesis in rats combined with seizure score monitoring to evaluate the antiepileptogenic effect of sarcosine. We used immunohistochemistry and Western blot analysis for the evaluation of GlyT1 expression and epigenetic changes of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in the epileptogenic hippocampi of rats, and further evaluated expression changes in enzymes involved in the regulation of DNA methylation, ten-eleven translocation methylcytosine dioxygenase 1 (TET1), DNA-methyltransferase 1 (DNMT1), and DNMT3a. Our results demonstrated: (i) experimental evidence that sarcosine (3 g/kg, i.p. daily) suppressed kindling epileptogenesis in rats; (ii) the sarcosine-induced antiepileptogenic effect was accompanied by a suppressed hippocampal GlyT1 expression as well as a reduction of hippocampal 5mC levels and a corresponding increase in 5hmC; and (iii) sarcosine treatment caused differential expression changes of TET1 and DNMTs. Together, these findings suggest that sarcosine has unprecedented disease-modifying properties in a kindling model of epileptogenesis in rats, which was associated with altered hippocampal DNA methylation. Thus, manipulation of the glycine system is a potential therapeutic approach to attenuate the development of epilepsy.
PubMed: 32581708
DOI: 10.3389/fnmol.2020.00097 -
The Journal of Nutrition Aug 2022Parenteral nutrition (PN) is often a necessity for preterm infants; however, prolonged PN leads to gut atrophy, weakened gut barrier function, and a higher risk of...
BACKGROUND
Parenteral nutrition (PN) is often a necessity for preterm infants; however, prolonged PN leads to gut atrophy, weakened gut barrier function, and a higher risk of intestinal infections. Peptide transporter-1 (PepT1) is a di- or tripeptide transporter in the gut and, unlike other nutrient transporters, its activity is preserved with the onset of intestinal atrophy from PN. As such, enteral amino acids in the form of dipeptides may be more bioavailable than free amino acids when atrophy is present.
OBJECTIVES
In Yucatan miniature piglets with PN-induced intestinal atrophy, we sought to determine the structural and functional effects of enteral refeeding with lysine as a dipeptide, compared to free L-lysine.
METHODS
Piglets aged 7-8 days were PN-fed for 4 days to induce intestinal atrophy, then were refed with enteral diets with equimolar lysine supplied as lysyl-lysine (Lys-Lys; n = 7), free lysine (n = 7), or Lys-Lys with glycyl-sarcosine (n = 6; to determine whether competitive inhibition of Lys-Lys uptake would abolish PepT1-mediated effects). The diets provided lysine at 75% of the requirement and were gastrically delivered for a total of 18 hours. Whole-body and tissue-specific protein synthesis, as well as indices for gut structure and barrier function, were measured.
RESULTS
The villus height, mucosal weight, and free lysine concentration were higher in the Lys-Lys group compared to the other 2 groups (P < 0.05). Lysyl-lysine led to greater whole-body protein synthesis compared to free lysine (P < 0.05). Mucosal myeloperoxidase activity was lower in the Lys-Lys group (P < 0.05), suggesting less inflammation. The inclusion of glycyl-sarcosine with Lys-Lys abolished the dipeptide effects on whole-body and tissue-specific protein synthesis (P < 0.05), suggesting that improved lysine availability was mediated by PepT1.
CONCLUSIONS
Improved intestinal structure and whole-body protein synthesis suggests that feeding strategies designed to exploit PepT1 may help to avoid adverse effects when enteral nutrition is reintroduced into the compromised guts of neonatal piglets.
Topics: Amino Acids; Animals; Atrophy; Dipeptides; Humans; Infant, Newborn; Infant, Premature; Intestinal Mucosa; Lysine; Sarcosine; Swine
PubMed: 35481706
DOI: 10.1093/jn/nxac095 -
Journal of Translational Medicine Nov 2023The morbidity of cancer keeps growing worldwide, and among that, the colorectal cancer (CRC) has jumped to third. Existing early screening tests for CRC are limited. The...
BACKGROUND
The morbidity of cancer keeps growing worldwide, and among that, the colorectal cancer (CRC) has jumped to third. Existing early screening tests for CRC are limited. The aim of this study was to develop a diagnostic strategy for CRC by plasma metabolomics.
METHODS
A targeted amino acids metabolomics method was developed to quantify 32 plasma amino acids in 130 CRC patients and 216 healthy volunteers, to identify potential biomarkers for CRC, and an independent sample cohort comprising 116 CRC subjects, 33 precancerosiss patients and 195 healthy volunteers was further used to validate the diagnostic model. Amino acids-related genes were retrieved from Gene Expression Omnibus and Molecular Signatures Database and analyzed.
RESULTS
Three were chosen out of the 32 plasma amino acids examined. The tryptophan / sarcosine / glutamic acid -based receiver operating characteristic (ROC) curve showed the area under the curve (AUC) of 0.955 (specificity 83.3% and sensitivity 96.8%) for all participants, and the logistic regression model were used to distinguish between early stage (I and II) of CRC and precancerosiss patients, which showed superiority to the commonly used carcinoembryonic antigen. The GO and KEGG enrichment analysis proved many alterations in amino acids metabolic pathways in tumorigenesis.
CONCLUSION
This altered plasma amino acid profile could effectively distinguish CRC patients from precancerosiss patients and healthy volunteers with high accuracy. Prognostic tests based on the tryptophan/sarcosine/glutamic acid biomarkers in the large population could assess the clinical significance of CRC early detection and intervention.
Topics: Humans; Amino Acids; Tryptophan; Sarcosine; Biomarkers, Tumor; Metabolomics; Colorectal Neoplasms; Glutamates
PubMed: 37978537
DOI: 10.1186/s12967-023-04604-7 -
International Journal of Molecular... Nov 2022Details on the unexpected formation of two new (dimethylamino)methyl corrole isomers from the reaction of 5,10,15-tris(pentafluorophenyl)corrolatogallium(III) with...
Details on the unexpected formation of two new (dimethylamino)methyl corrole isomers from the reaction of 5,10,15-tris(pentafluorophenyl)corrolatogallium(III) with sarcosine and paraformaldehyde are presented. Semi-empirical calculations on possible mechanism pathways seem to indicate that the new compounds are probably formed through a Mannich-type reaction. The extension of the protocol to the free-base 5,10,15-tris(pentafluorophenyl)corrole afforded an unexpected new seven-membered ring corrole derivative, confirming the peculiar behavior of corroles towards known reactions when compared to the well-behaved porphyrin counterparts.
Topics: Sarcosine; Porphyrins; Isomerism
PubMed: 36362367
DOI: 10.3390/ijms232113581 -
Asia Pacific Journal of Clinical... 2022To optimize the pretreatment method of colorectal cancer tissue samples for metabolomics research based on solid-phase nuclear magnetic resonance (NMR).
BACKGROUND AND OBJECTIVES
To optimize the pretreatment method of colorectal cancer tissue samples for metabolomics research based on solid-phase nuclear magnetic resonance (NMR).
METHODS AND STUDY DESIGN
The mucosal tissues of colorectal cancer were classified into five groups with a volume of 0.2 cm*0.2 cm*0.2 cm. The pretreatment methods for each group were as follows: I. Preservation with liquid nitrogen alone. Samples were also treated with liquid nitrogen for 10 (II), 20 (III), and 30 min (IV), respectively, immediately after isolation and then transferred to a -80℃ refrigerator; V. Only -80℃ refrigerator storage. No more than 30 minutes should pass between isolation and pretreatment of tumor samples. The tissue sample testing process was carried out on Bruker AVII-600 NMR Spectrometer. NMR signals were collected and analysed using partial least-squares discrimination analysis (PLS-DA) to explore the effects of different pretreatment methods on the metabolic changes of samples.
RESULTS
The levels of pelargonic acid, stearic acid, D-Ribose, heptadecanoic acid, pyruvic acid, succinate, sarcosine, glycine, creatine, and L-lactate in the group I (only liquid nitrogen) were significantly lower than the other groups (p<0.05); the content of glycerophosphocholine in the group I (only liquid nitrogen) was lower than that in the other groups (p=0.055). These indicated that the glucose and choline phospholipid metabolism levels of the liquid nitrogen group were significantly lower than those of the other four groups.
CONCLUSIONS
Liquid nitrogen storage can stop the metabolic process of glucose and choline phospholipid in colorectal cancer tissue samples in vitro, thus maintaining the metabolic state of tissue samples in vivo as much as possible.
Topics: Choline; Colorectal Neoplasms; Creatine; Glucose; Glycerylphosphorylcholine; Humans; Lactates; Nitrogen; Pyruvic Acid; Ribose; Sarcosine; Succinates
PubMed: 36173224
DOI: 10.6133/apjcn.202209_31(3).0020