Promiscuous enzymes generating d-amino acids in mammals: Why they may still surprise us?

2021 ◽  
Vol 478 (5) ◽  
pp. 1175-1178
Author(s):  
Herman Wolosker ◽  
Inna Radzishevsky
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Common Property ◽  
Mammalian Brain ◽  
Intercellular Signaling ◽  
Biochemical Pathway ◽  
Recent Issue ◽  
Threonine Dehydratase ◽  
Glutamate Synthesis ◽  
Potential Biomarkers

Promiscuous catalysis is a common property of enzymes, particularly those using pyridoxal 5′-phosphate as a cofactor. In a recent issue of this journal, Katane et al. Biochem. J. 477, 4221–4241 demonstrate the synthesis and accumulation of d-glutamate in mammalian cells by promiscuous catalysis mediated by a pyridoxal 5′-phosphate enzyme, the serine/threonine dehydratase-like (SDHL). The mechanism of SDHL resembles that of serine racemase, which synthesizes d-serine, a well-established signaling molecule in the mammalian brain. d-Glutamate is present in body fluids and is degraded by the d-glutamate cyclase at the mitochondria. This study demonstrates a biochemical pathway for d-glutamate synthesis in mammalian cells and advances our knowledge on this little-studied d-amino acid in mammals. d-Amino acids may still surprise us by their unique roles in biochemistry, intercellular signaling, and as potential biomarkers of disease.

scholarly journals Identification of an l-serine/l-threonine dehydratase with glutamate racemase activity in mammals

2020 ◽  
Vol 477 (21) ◽  
pp. 4221-4241
Author(s):  
Masumi Katane ◽  
Kento Nakasako ◽  
Kanato Yako ◽  
Yasuaki Saitoh ◽  
Masae Sekine ◽  
...  
Keyword(s):  
Amino Acids ◽  
Metabolic Pathways ◽  
Mammalian Cells ◽  
Biosynthetic Pathway ◽  
Threonine Dehydratase ◽  
Glutamate Racemase ◽  
Mammalian Tissues ◽  
Serine Dehydratase ◽  
Degradative Enzyme ◽  
Dehydratase Activity

Recent investigations have shown that multiple d-amino acids are present in mammals and these compounds have distinctive physiological functions. Free d-glutamate is present in various mammalian tissues and cells and in particular, it is presumably correlated with cardiac function, and much interest is growing in its unique metabolic pathways. Recently, we first identified d-glutamate cyclase as its degradative enzyme in mammals, whereas its biosynthetic pathway in mammals is unclear. Glutamate racemase is a most probable candidate, which catalyzes interconversion between d-glutamate and l-glutamate. Here, we identified the cDNA encoding l-serine dehydratase-like (SDHL) as the first mammalian clone with glutamate racemase activity. This rat SDHL had been deposited in mammalian databases as a protein of unknown function and its amino acid sequence shares ∼60% identity with that of l-serine dehydratase. Rat SDHL was expressed in Escherichia coli, and the enzymatic properties of the recombinant were characterized. The results indicated that rat SDHL is a multifunctional enzyme with glutamate racemase activity in addition to l-serine/l-threonine dehydratase activity. This clone is hence abbreviated as STDHgr. Further experiments using cultured mammalian cells confirmed that d-glutamate was synthesized and l-serine and l-threonine were decomposed. It was also found that SDHL (STDHgr) contributes to the homeostasis of several other amino acids.

scholarly journals Metabolic Alterations Associated with γ-Hydroxybutyric Acid and the Potential of Metabolites as Biomarkers of Its Exposure

Metabolites ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 101
Author(s):  
Suryun Jung ◽  
Suji Kim ◽  
Yujin Seo ◽  
Sooyeun Lee
Keyword(s):  
Amino Acids ◽  
Biological Samples ◽  
Metabolic Pathways ◽  
Short Chain Fatty Acid ◽  
Chain Fatty Acid ◽  
Mammalian Brain ◽  
Clinical Settings ◽  
Hydroxybutyric Acid ◽  
Metabolic Alterations ◽  
Potential Biomarkers

γ-Hydroxybutyric acid (GHB) is an endogenous short chain fatty acid that acts as a neurotransmitter and neuromodulator in the mammalian brain. It has often been illegally abused or misused due to its strong anesthetic effect, particularly in drug-facilitated crimes worldwide. However, proving its ingestion is not straightforward because of the difficulty in distinguishing between endogenous and exogenous GHB, as well as its rapid metabolism. Metabolomics and metabolism studies have recently been used to identify potential biomarkers of GHB exposure. This mini-review provides an overview of GHB-associated metabolic alterations and explores the potential of metabolites for application as biomarkers of GHB exposure. For this, we discuss the biosynthesis and metabolism of GHB, analytical issues of GHB in biological samples, alterations in metabolic pathways, and changes in the levels of GHB conjugates in biological samples from animal and human studies. Metabolic alterations in organic acids, amino acids, and polyamines in urine enable discrimination between GHB-ingested animals or humans and controls. The potential of GHB conjugates has been investigated in a variety of clinical settings. Despite the recent growth in the application of metabolomics and metabolism studies associated with GHB exposure, it remains challenging to distinguish between endogenous and exogenous GHB. This review highlights the significance of further metabolomics and metabolism studies for the discovery of practical peripheral biomarkers of GHB exposure.

scholarly journals Autophagy Deficiency by Atg4B Loss Leads to Metabolomic Alterations in Mice

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 481
Author(s):  
Gemma G. Martínez-García ◽  
Raúl F. Pérez ◽  
Álvaro F. Fernández ◽  
Sylvere Durand ◽  
Guido Kroemer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Mammalian Cells ◽  
Tissue Homeostasis ◽  
In Vivo Studies ◽  
Protective Mechanism ◽  
Cardiac Damage ◽  
Wide Range ◽  
First Time

Autophagy is an essential protective mechanism that allows mammalian cells to cope with a variety of stressors and contributes to maintaining cellular and tissue homeostasis. Due to these crucial roles and also to the fact that autophagy malfunction has been described in a wide range of pathologies, an increasing number of in vivo studies involving animal models targeting autophagy genes have been developed. In mammals, total autophagy inactivation is lethal, and constitutive knockout models lacking effectors of this route are not viable, which has hindered so far the analysis of the consequences of a systemic autophagy decline. Here, we take advantage of atg4b−/− mice, an autophagy-deficient model with only partial disruption of the process, to assess the effects of systemic reduction of autophagy on the metabolome. We describe for the first time the metabolic footprint of systemic autophagy decline, showing that impaired autophagy results in highly tissue-dependent alterations that are more accentuated in the skeletal muscle and plasma. These changes, which include changes in the levels of amino-acids, lipids, or nucleosides, sometimes resemble those that are frequently described in conditions like aging, obesity, or cardiac damage. We also discuss different hypotheses on how impaired autophagy may affect the metabolism of several tissues in mammals.

scholarly journals Amino Acids Rather than Glucose Account for the Majority of Cell Mass in Proliferating Mammalian Cells

2016 ◽  
Vol 36 (5) ◽  
pp. 540-549 ◽  
Author(s):  
Aaron M. Hosios ◽  
Vivian C. Hecht ◽  
Laura V. Danai ◽  
Marc O. Johnson ◽  
Jeffrey C. Rathmell ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Cell Mass

scholarly journals Finding the Binding Site of Peloruside A and its Secondary Effects in Saccharomyces Cerevisiae using a  Chemical Genetics Approach

2021 ◽  
Author(s):  
◽  
Reem Hanna
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Amino Acids ◽  
Flow Cytometry ◽  
Binding Site ◽  
Mammalian Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Peloruside A ◽  
Microtubule Function

<p>Peloruside A, a natural product isolated from the marine sponge Mycale hentscheli, is a microtubule-stabilising agent that has a similar mechanism of action to the anticancer drug paclitaxel and is cytotoxic to cultured mammalian cells. Peloruside appears to bind to a distinct site on mammalian tubulin that is different from that of the taxoid-site drugs. Because of the high sequence homology between yeast and mammalian tubulin, Saccharomyces cerevisiae (S. cerevisiae) was used as a model organism to characterise the peloruside-binding site with the aim of advancing our understanding about this site on mammalian tubulin. Wild type S. cerevisiae (BY4741) was sensitive to peloruside at uM concentrations; however, a strain that lacks the mad2 (Mitotic Arrest Deficient 2) gene showed increased sensitivity to the drug at much lower uM concentrations. This gene is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. The main aims of this project were to define the binding site of peloruside A using yeast tubulin to see if microtubule function and/or morphology is altered in yeast by peloruside, and to identify any secondary drug targets "friends of the target" through chemical genetic interactions profiling (Homozygous deletion profiling microarray). Site-directed mutagenesis was used to mutate two conserved amino acids (A296T; R306H) known to confer resistance to peloruside in mammalian cells. Based on a published computer model of the peloruside binding site on mammalian tubulin, we also mutated three other amino acids, two that were predicted to affect peloruside binding (Q291M and N337L), and one that was predicted to affect laulimalide binding but have little affect on peloruside binding (V333W). We also included a negative control that was predicted to have no effect on peloruside binding (R282Q) and would affect epothilone binding. We found that of the six point mutations, only Q291M failed to confer resistance in yeast and instead it increased the inhibition to the drug. Using a bud index assay, confocal microscopy, and flow cytometry, 40-50 uM peloruside was shown to block cells in G2/M of the cell cycle, confirming a direct action of the drug on microtubule function. Homozygous profiling (HOP) microarray analysis of a deletion mutant set of yeast genes was also carried out to identify gene products that interact with peloruside in order to link the drug to specific networks or biochemical pathways in the cells. From site-directed mutagenesis, we concluded that peloruside binds to yeast B-tubulin in the region predicted by the published model of the binding site, and therefore mapping the site on yeast tubulin could provide useful information about the mammalian binding site for peloruside. The bud index, flow cytometry, and confocal microscopy experiments provided further evidence that peloruside interacts with yeast tubulin. From HOP we found that peloruside has roles in the cell cycle, as expected, and has effects on protein transport, secretion, cell wall synthesis, and steroid biosynthesis pathways.</p>

scholarly journals A Genetically Encoded Picolyl Azide for Improved Live Cell Copper Click Labeling

2021 ◽  
Vol 9 ◽  
Author(s):  
Birthe Meineke ◽  
Johannes Heimgärtner ◽  
Alexander J. Craig ◽  
Michael Landreh ◽  
Lindon W. K. Moodie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Stop Codon ◽  
Bioorthogonal Chemistry ◽  
Bioorthogonal Reaction ◽  
Noncanonical Amino Acids ◽  
Selective Reactivity ◽  
Direct Incorporation ◽  
Chelating Groups ◽  
Amber Suppression

Bioorthogonal chemistry allows rapid and highly selective reactivity in biological environments. The copper-catalyzed azide–alkyne cycloaddition (CuAAC) is a classic bioorthogonal reaction routinely used to modify azides or alkynes that have been introduced into biomolecules. Amber suppression is an efficient method for incorporating such chemical handles into proteins on the ribosome, in which noncanonical amino acids (ncAAs) are site specifically introduced into the polypeptide in response to an amber (UAG) stop codon. A variety of ncAA structures containing azides or alkynes have been proven useful for performing CuAAC chemistry on proteins. To improve CuAAC efficiency, biologically incorporated alkyne groups can be reacted with azide substrates that contain copper-chelating groups. However, the direct incorporation of copper-chelating azides into proteins has not been explored. To remedy this, we prepared the ncAA paz-lysine (PazK), which contains a picolyl azide motif. We show that PazK is efficiently incorporated into proteins by amber suppression in mammalian cells. Furthermore, PazK-labeled proteins show improved reactivity with alkyne reagents in CuAAC.

scholarly journals Homeostasis of branched-chain amino acids is critical for the activity of TOR signaling in Arabidopsis

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Pengfei Cao ◽  
Sang-Jin Kim ◽  
Anqi Xing ◽  
Craig A Schenck ◽  
Lu Liu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Branched Chain Amino Acids ◽  
Nutrient Sensing ◽  
Tor Signaling ◽  
Functional Connection ◽  
Primary Input ◽  
Tor Kinase ◽  
Arabidopsis Mutant ◽  
Branched Chain

The target of rapamycin (TOR) kinase is an evolutionarily conserved hub of nutrient sensing and metabolic signaling. In plants, a functional connection of TOR activation with glucose availability was demonstrated, while it is yet unclear whether branched-chain amino acids (BCAAs) are a primary input of TOR signaling as they are in yeast and mammalian cells. Here, we report on the characterization of an Arabidopsis mutant over-accumulating BCAAs. Through chemical interventions targeting TOR and by examining mutants of BCAA biosynthesis and TOR signaling, we found that BCAA over-accumulation leads to up-regulation of TOR activity, which causes reorganization of the actin cytoskeleton and actin-associated endomembranes. Finally, we show that activation of TOR is concomitant with alteration of cell expansion, proliferation and specialized metabolism, leading to pleiotropic effects on plant growth and development. These results demonstrate that BCAAs contribute to plant TOR activation and reveal previously uncharted downstream subcellular processes of TOR signaling.

scholarly journals The GC-TOF/MS-based Metabolomic Analysis Reveals Altered Metabolic Profiles in Nitrogen-Deficient Leaves and Roots of Tea Plants (Camellia sinensis L.)

2021 ◽  
Author(s):  
Zheng-He Lin ◽  
Chang-Song Chen ◽  
Qiu-Sheng Zhong ◽  
Qi-Chun Ruan ◽  
Zhi-Hui Chen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Organic Acids ◽  
N Fertilizer ◽  
Metabolic Profiles ◽  
Tea Leaves ◽  
N Deficiency ◽  
Leaves And Roots ◽  
Tea Plants ◽  
Potential Biomarkers ◽  
Tof Ms

Abstract Background: Nitrogen (N) fertilizer is commonly considered as one of the most omportant limiting factors in the agricultural production. As a result, modern tea production, a large amount of N fertilizer is used to improve the yield. Unfortunately, the large amount of N fertilizer input has led to increased plant nitrogen-tolerance and decreased amplitude of yield improvement, which results in significant N loss, energy waste and environment pollution.However, the effects of N-deficiency on the metabolic profiles of leaves and roots are not well understood.Results: In the study, seedlings of Camellia sinensis (L.) O. Kuntze cv. Chunlv 2 were treated with 3 mM NH4NO3（as Control）or without NH4NO3（as N-deficiency）for 4 months by sandy culture. The results suggested the N-deficiency induced tea leaf chlorosis, impaired biomass accumulation, decreased the leaf chlorophyll content and N absorption compared to Control. The untargeted metabolomics based on GC-TOF/MS approach revealed discrimination of the metabolic profiles between N-deficient tea leaves and roots. The identification and classification of the altered metabolites indicated the N deficiency upregulated the relative abundances of most phenylpropanoids, organic acids while downregulated the relative abundances of most amino acids in the tea leaves. Differentially, N-deficiency induced the accumulation of most carbohydrates, organic acids and amino acids in the tea roots. The potential biomarkers screened in the N-deficient leaves compared to Control reflected the N deficiency reduced the tea quality. Unlike the N-deficient leaves, the potential biomarkers in the N-deficient roots implied an improved stress response. Conclusions:The results demonstrated the N deficiency had different effects on the primary and secondary metabolic alteration of tea leaves and roots. The findings of the study will facilitate a comprehensive understanding of the N-deficient tea plants and provide a valuable reference for the optimized N nutrient management in the tea plantations.

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