scholarly journals Chlamydia trachomatis dapFEncodes a Bifunctional Enzyme Capable of Both D-Glutamate Racemase and Diaminopimelate Epimerase Activities

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
George Liechti ◽  
Raghuveer Singh ◽  
Patricia L. Rossi ◽  
Miranda D. Gray ◽  
Nancy E. Adams ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chlamydia Trachomatis ◽  
Bacterial Infections ◽  
Pyridoxal Phosphate ◽  
De Novo ◽  
Bacterial Species ◽  
Genetic Complementation ◽  
Enzyme Promiscuity ◽  
Peptidoglycan Synthesis ◽  
Glutamate Racemase

ABSTRACTPeptidoglycan is a sugar/amino acid polymer unique to bacteria and essential for division and cell shape maintenance. Thed-amino acids that make up its cross-linked stem peptides are not abundant in nature and must be synthesized by bacteriade novo.d-Glutamate is present at the second position of the pentapeptide stem and is strictly conserved in all bacterial species. In Gram-negative bacteria,d-glutamate is generated via the racemization ofl-glutamate by glutamate racemase (MurI).Chlamydia trachomatisis the leading cause of infectious blindness and sexually transmitted bacterial infections worldwide. While its genome encodes a majority of the enzymes involved in peptidoglycan synthesis, nomurIhomologue has ever been annotated. Recent studies have revealed the presence of peptidoglycan inC. trachomatisand confirmed that its pentapeptide includesd-glutamate. In this study, we show thatC. trachomatissynthesizesd-glutamate by utilizing a novel, bifunctional homologue of diaminopimelate epimerase (DapF). DapF catalyzes the final step in the synthesis ofmeso-diaminopimelate, another amino acid unique to peptidoglycan. Genetic complementation of anEscherichia coli murImutant demonstrated thatChlamydiaDapF can generated-glutamate. Biochemical analysis showed robust activity, but unlike canonical glutamate racemases, activity was dependent on the cofactor pyridoxal phosphate. Genetic complementation, enzymatic characterization, and bioinformatic analyses indicate that chlamydial DapF shares characteristics with other promiscuous/primordial enzymes, presenting a potential mechanism ford-glutamate synthesis not only inChlamydiabut also numerous other genera within thePlanctomycetes-Verrucomicrobiae-Chlamydiaesuperphylum that lack recognized glutamate racemases.IMPORTANCEHere we describe one of the last remaining “missing” steps in peptidoglycan synthesis in pathogenicChlamydiaspecies, the synthesis ofd-glutamate. We have determined that the diaminopimelate epimerase (DapF) encoded byChlamydia trachomatisis capable of carrying out both the epimerization of DAP and the pyridoxal phosphate-dependent racemization of glutamate. Enzyme promiscuity is thought to be the hallmark of early microbial life on this planet, and there is currently an active debate as to whether “moonlighting enzymes” represent primordial evolutionary relics or are a product of more recent reductionist evolutionary pressures. Given the large number ofChlamydiaspecies (as well as members of thePlanctomycetes-Verrucomicrobiae-Chlamydiaesuperphylum) that possess DapF but lack homologues of MurI, it is likely that DapF is a primordial isomerase that functions as both racemase and epimerase in these organisms, suggesting that specializedd-glutamate racemase enzymes never evolved in these microbes.

scholarly journals Computational evidence of a new allosteric communication pathway between active sites and putative regulatory sites in the alanine racemase ofMycobacterium tuberculosis

2018 ◽  
Author(s):  
Jayanthy Jyothikumar ◽  
Sushil Chandani ◽  
Tangirala Ramakrishna
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Discovery ◽  
Side Effects ◽  
Active Site ◽  
Bacterial Infections ◽  
Active Sites ◽  
Pyridoxal Phosphate ◽  
Bacterial Species ◽  
Alanine Racemase ◽  
Docking Simulations

AbstractAlanine racemase, a popular drug target fromMycobacterium tuberculosis, catalyzes the biosynthesis of D-alanine, an essential component in bacterial cell walls. With the help of elastic network models of alanine racemase fromMycobacterium tuberculosis, we show that the mycobacterial enzyme fluctuates between two undiscovered states—a closed and an open state. A previous experimental screen identified several drug-like lead compounds against the mycobacterial alanine racemase, whose inhibitory mechanisms are not known. Docking simulations of the inhibitor leads onto the mycobacterial enzyme conformations obtained from the dynamics of the enzyme provide first clues to a putative regulatory role for two new pockets targeted by the leads. Further, our results implicate the movements of a short helix, behind the communication between the new pockets and the active site, indicating allosteric mechanisms for the inhibition. Based on our findings, we theorize that catalysis is feasible only in the open state. The putative regulatory pockets and the enzyme fluctuations are conserved across several alanine racemase homologs from diverse bacterial species, mostly pathogenic, pointing to a common regulatory mechanism important in drug discovery.Author summaryIn spite of the discovery of many inhibitors against the TB-causing pathogenMycobacterium tuberculosis, only a very few have reached the market as effective TB drugs. Most of the marketed TB drugs induce toxic side effects in patients, as they non-specifically target human cells in addition to pathogens. One such TB drug, D-cycloserine, targets pyridoxal phosphate moiety non-specifically regardless of whether it is present in the pathogen or the human host enzymes. D-cycloserine was developed to inactivate alanine racemase in TB causing pathogen. Alanine racemase is a bacterial enzyme essential in cell wall synthesis. Serious side effects caused by TB drugs like D-cycloserine, lead to patients’ non-compliance with treatment regimen, often causing fatal outcomes. Current drug discovery efforts focus on finding specific, non-toxic TB drugs. Through computational studies, we have identified new pockets on the mycobacterial alanine racemase and show that they can bind drug-like compounds. The location of these pockets away from the pyridoxal phosphate-containing active site, make them attractive target sites for novel, specific TB drugs. We demonstrate the presence of these pockets in alanine racemases from several pathogens and expect our findings to accelerate the discovery of non-toxic drugs against TB and other bacterial infections.

Reaction Mechanism of Glutamate Racemase, a Pyridoxal Phosphate-Independent Amino Acid Racemase

1992 ◽  
Vol 112 (1) ◽  
pp. 139-142 ◽  
Author(s):  
Soo-Young Choi ◽  
Nobuyoshi Esaki ◽  
Tohru Yoshimura ◽  
Kenji Soda
Keyword(s):  
Reaction Mechanism ◽  
Amino Acid ◽  
Pyridoxal Phosphate ◽  
Glutamate Racemase ◽  
Amino Acid Racemase

Fluoroquinolone-3-carboxamide Amino Acid Conjugates: Synthesis, Antibacterial Properties And Molecular Modeling Studies

2020 ◽  
Vol 17 (1) ◽  
pp. 71-84
Author(s):  
Riham M. Bokhtia ◽  
Siva S. Panda ◽  
Adel S. Girgis ◽  
Hitesh H. Honkanadavar ◽  
Tarek S. Ibrahim ◽  
...  
Keyword(s):  
Experimental Data ◽  
Antibacterial Activity ◽  
Amino Acid ◽  
Bacterial Infections ◽  
Antibacterial Agents ◽  
Antibacterial Properties ◽  
Computational Studies ◽  
Protecting Group ◽  
Amino Acid Conjugates ◽  
Molecular Modeling Studies

Background: Bacterial infections are considered as one of the major global health threats, so it is very essential to design and develop new antibacterial agents to overcome the drawbacks of existing antibacterial agents. Method: The aim of this work is to synthesize a series of new fluoroquinolone-3-carboxamide amino acid conjugates by molecular hybridization. We utilized benzotriazole chemistry to synthesize the desired hybrid conjugates. Result: All the conjugates were synthesized in good yields, characterized, evaluated for their antibacterial activity. The compounds were screened for their antibacterial activity using methods adapted from the Clinical and Laboratory Standards Institute. Synthesized conjugates were tested for activity against medically relevant pathogens; Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27856) Staphylococcus aureus (ATCC 25923) and Enterococcus faecalis (ATCC 19433). Conclusion: The observed antibacterial experimental data indicates the selectivity of our synthesized conjugates against E.Coli. The protecting group on amino acids decreases the antibacterial activity. The synthesized conjugates are non-toxic to the normal cell lines. The experimental data were supported by computational studies.

Amino Acids Sequence-based Analysis of Arginine Deiminase from Different Prokaryotic Organisms: An In Silico Approach

2020 ◽  
Vol 14 (3) ◽  
pp. 235-246
Author(s):  
Sara Abdollahi ◽  
Mohammad H. Morowvat ◽  
Amir Savardashtaki ◽  
Cambyz Irajie ◽  
Sohrab Najafipour ◽  
...  
Keyword(s):  
Amino Acid ◽  
Physicochemical Properties ◽  
Sequence Alignment ◽  
Multiple Sequence Alignment ◽  
In Silico ◽  
Bacterial Species ◽  
Arginine Deiminase ◽  
Antitumor Effects ◽  
Multiple Sequence ◽  
In Silico Studies

Background: Arginine deiminase is a bacterial enzyme, which degrades L-arginine. Some human cancers such as hepatocellular carcinoma (HCC) and melanoma are auxotrophic for arginine. Therefore, PEGylated arginine deiminase (ADI-PEG20) is a good anticancer candidate with antitumor effects. It causes local depletion of L-arginine and growth inhibition in arginineauxotrophic tumor cells. The FDA and EMA have granted orphan status to this drug. Some recently published patents have dealt with this enzyme or its PEGylated form. Objective: Due to increasing attention to it, we aimed to evaluate and compare 30 arginine deiminase proteins from different bacterial species through in silico analysis. Methods: The exploited analyses included the investigation of physicochemical properties, multiple sequence alignment (MSA), motif, superfamily, phylogenetic and 3D comparative analyses of arginine deiminase proteins thorough various bioinformatics tools. Results: The most abundant amino acid in the arginine deiminase proteins is leucine (10.13%) while the least amino acid ratio is cysteine (0.98%). Multiple sequence alignment showed 47 conserved patterns between 30 arginine deiminase amino acid sequences. The results of sequence homology among 30 different groups of arginine deiminase enzymes revealed that all the studied sequences located in amidinotransferase superfamily. Based on the phylogenetic analysis, two major clusters were identified. Considering the results of various in silico studies; we selected the five best candidates for further investigations. The 3D structures of the best five arginine deiminase proteins were generated by the I-TASSER server and PyMOL. The RAMPAGE analysis revealed that 81.4%-91.4%, of the selected sequences, were located in the favored region of arginine deiminase proteins. Conclusion: The results of this study shed light on the basic physicochemical properties of thirty major arginine deiminase sequences. The obtained data could be employed for further in vivo and clinical studies and also for developing the related therapeutic enzymes.

scholarly journals Discovery of novel community-relevant small proteins in a simplified human intestinal microbiome

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Hannes Petruschke ◽  
Christian Schori ◽  
Sebastian Canzler ◽  
Sarah Riesbeck ◽  
Anja Poehlein ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Intestinal Microbiota ◽  
De Novo ◽  
Bacterial Species ◽  
Intestinal Microbiome ◽  
Single Strain ◽  
Small Proteins ◽  
Human Intestinal Microbiota ◽  
Wide Range

Abstract Background The intestinal microbiota plays a crucial role in protecting the host from pathogenic microbes, modulating immunity and regulating metabolic processes. We studied the simplified human intestinal microbiota (SIHUMIx) consisting of eight bacterial species with a particular focus on the discovery of novel small proteins with less than 100 amino acids (= sProteins), some of which may contribute to shape the simplified human intestinal microbiota. Although sProteins carry out a wide range of important functions, they are still often missed in genome annotations, and little is known about their structure and function in individual microbes and especially in microbial communities. Results We created a multi-species integrated proteogenomics search database (iPtgxDB) to enable a comprehensive identification of novel sProteins. Six of the eight SIHUMIx species, for which no complete genomes were available, were sequenced and de novo assembled. Several proteomics approaches including two earlier optimized sProtein enrichment strategies were applied to specifically increase the chances for novel sProtein discovery. The search of tandem mass spectrometry (MS/MS) data against the multi-species iPtgxDB enabled the identification of 31 novel sProteins, of which the expression of 30 was supported by metatranscriptomics data. Using synthetic peptides, we were able to validate the expression of 25 novel sProteins. The comparison of sProtein expression in each single strain versus a multi-species community cultivation showed that six of these sProteins were only identified in the SIHUMIx community indicating a potentially important role of sProteins in the organization of microbial communities. Two of these novel sProteins have a potential antimicrobial function. Metabolic modelling revealed that a third sProtein is located in a genomic region encoding several enzymes relevant for the community metabolism within SIHUMIx. Conclusions We outline an integrated experimental and bioinformatics workflow for the discovery of novel sProteins in a simplified intestinal model system that can be generically applied to other microbial communities. The further analysis of novel sProteins uniquely expressed in the SIHUMIx multi-species community is expected to enable new insights into the role of sProteins on the functionality of bacterial communities such as those of the human intestinal tract.

scholarly journals Amino Acid Transporter LAT1 (SLC7A5) Mediates MeHg-Induced Oxidative Stress Defense in the Human Placental Cell Line HTR-8/SVneo

2021 ◽  
Vol 22 (4) ◽  
pp. 1707
Author(s):  
Sebastian Granitzer ◽  
Raimund Widhalm ◽  
Martin Forsthuber ◽  
Isabella Ellinger ◽  
Gernot Desoye ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Amino Acid ◽  
De Novo ◽  
Structural Similarity ◽  
Amino Acid Transporter ◽  
Cell Number ◽  
Mehg Exposure ◽  
Acid Transporter ◽  
And Oxidative Stress

The placental barrier can protect the fetus from contact with harmful substances. The potent neurotoxin methylmercury (MeHg), however, is very efficiently transported across the placenta. Our previous data suggested that L-type amino acid transporter (LAT)1 is involved in placental MeHg uptake, accepting MeHg-L-cysteine conjugates as substrate due to structural similarity to methionine. The aim of the present study was to investigate the antioxidant defense of placental cells to MeHg exposure and the role of LAT1 in this response. When trophoblast-derived HTR-8/SVneo cells were LAT1 depleted by siRNA-mediated knockdown, they accumulated less MeHg. However, they were more susceptible to MeHg-induced toxicity. This was evidenced in decreased cell viability at a usually noncytotoxic concentration of 0.03 µM MeHg (~6 µg/L). Treatment with ≥0.3 µM MeHg increased cytotoxicity, apoptosis rate, and oxidative stress of HTR-8/SVneo cells. These effects were enhanced under LAT1 knockdown. Reduced cell number was seen when MeHg-exposed cells were cultured in medium low in cysteine, a constituent of the tripeptide glutathione (GSH). Because LAT1-deficient HTR-8/SVneo cells have lower GSH levels than control cells (independent of MeHg treatment), we conclude that LAT1 is essential for de novo synthesis of GSH, required to counteract oxidative stress. Genetic predisposition to decreased LAT1 function combined with MeHg exposure could increase the risk of placental damage.

scholarly journals The microbiota is dispensable for the early stages of peripheral regulatory T cell induction within mesenteric lymph nodes

2021 ◽  
Author(s):  
Carolin Wiechers ◽  
Mangge Zou ◽  
Eric Galvez ◽  
Michael Beckstette ◽  
Maria Ebel ◽  
...  
Keyword(s):  
T Cell ◽  
Lymph Nodes ◽  
Regulatory T Cell ◽  
De Novo ◽  
Bacterial Species ◽  
Short Chain Fatty Acids ◽  
Mesenteric Lymph Nodes ◽  
Direct Role ◽  
Early Stages ◽  
Mesenteric Lymph

AbstractIntestinal Foxp3+ regulatory T cell (Treg) subsets are crucial players in tolerance to microbiota-derived and food-borne antigens, and compelling evidence suggests that the intestinal microbiota modulates their generation, functional specialization, and maintenance. Selected bacterial species and microbiota-derived metabolites, such as short-chain fatty acids (SCFAs), have been reported to promote Treg homeostasis in the intestinal lamina propria. Furthermore, gut-draining mesenteric lymph nodes (mLNs) are particularly efficient sites for the generation of peripherally induced Tregs (pTregs). Despite this knowledge, the direct role of the microbiota and their metabolites in the early stages of pTreg induction within mLNs is not fully elucidated. Here, using an adoptive transfer-based pTreg induction system, we demonstrate that neither transfer of a dysbiotic microbiota nor dietary SCFA supplementation modulated the pTreg induction capacity of mLNs. Even mice housed under germ-free (GF) conditions displayed equivalent pTreg induction within mLNs. Further molecular characterization of these de novo induced pTregs from mLNs by dissection of their transcriptomes and accessible chromatin regions revealed that the microbiota indeed has a limited impact and does not contribute to the initialization of the Treg-specific epigenetic landscape. Overall, our data suggest that the microbiota is dispensable for the early stages of pTreg induction within mLNs.

scholarly journals Dietary Essential Amino Acid Restriction Promotes Hyperdipsia via Hepatic FGF21

Nutrients ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1469
Author(s):  
Patricia M. Rusu ◽  
Andrea Y. Chan ◽  
Mathias Heikenwalder ◽  
Oliver J. Müller ◽  
Adam J. Rose
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Dietary Protein ◽  
Essential Amino Acid ◽  
De Novo ◽  
Fibroblast Growth Factor 21 ◽  
Fibroblast Growth ◽  
De Novo Biosynthesis ◽  
Dietary Requirements ◽  
Dietary Amino Acid

Prior studies have reported that dietary protein dilution (DPD) or amino acid dilution promotes heightened water intake (i.e., hyperdipsia) however, the exact dietary requirements and the mechanism responsible for this effect are still unknown. Here, we show that dietary amino acid (AA) restriction is sufficient and required to drive hyperdipsia during DPD. Our studies demonstrate that particularly dietary essential AA (EAA) restriction, but not non-EAA, is responsible for the hyperdipsic effect of total dietary AA restriction (DAR). Additionally, by using diets with varying amounts of individual EAA under constant total AA supply, we demonstrate that restriction of threonine (Thr) or tryptophan (Trp) is mandatory and sufficient for the effects of DAR on hyperdipsia and that liver-derived fibroblast growth factor 21 (FGF21) is required for this hyperdipsic effect. Strikingly, artificially introducing Thr de novo biosynthesis in hepatocytes reversed hyperdipsia during DAR. In summary, our results show that the DPD effects on hyperdipsia are induced by the deprivation of Thr and Trp, and in turn, via liver/hepatocyte-derived FGF21.

Diagnostic tests for detecting Chlamydia trachomatis and Neisseria gonorrhoeae in rectal and pharyngeal specimens

2021 ◽  
Author(s):  
Paul C. Adamson ◽  
Jeffrey D. Klausner
Keyword(s):  
Chlamydia Trachomatis ◽  
Neisseria Gonorrhoeae ◽  
Bacterial Infections ◽  
Point Of Care ◽  
High Sensitivity ◽  
Population Level ◽  
The United States ◽  
Nucleic Acid Amplification ◽  
Point Of Care Test ◽  
Control And Prevention

Chlamydia trachomatis and Neisseria gonorrhoeae are two of the most often reported bacterial infections in the United States. The rectum and oropharynx are important anatomic sites of infection and can contribute to ongoing transmission. Nucleic acid amplification tests (NAATs) are the mainstays for the detection of C. trachomatis and N. gonorrhoeae infections owing to their high sensitivity and specificity. Several NAATs have been evaluated for testing in rectal and pharyngeal infections. A few assays recently received clearance by the Food and Drug Administration, including one point-of-care test. Those assays can be used for testing in symptomatic individuals, as well as for asymptomatic screening in certain patient populations. Routine screening for C. trachomatis in pharyngeal specimens is not recommended by the Centers for Disease Control and Prevention, though is often performed due to the use of multiplex assays. While expanding the types of settings for screening and using self-collected rectal and pharyngeal specimens can help to increase access and uptake of testing, additional research is needed to determine the potential benefits and costs associated with increased screening for rectal and pharyngeal C. trachomatis and N. gonorrhoeae infections on a population level.

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