α-Aminoadipate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2327-2334 ◽  
Author(s):  
Takashi Miyazaki ◽  
Junichi Miyazaki ◽  
Hisakazu Yamane ◽  
Makoto Nishiyama
Keyword(s):  
Thermus Thermophilus ◽  
Tca Cycle ◽  
Thermophilic Bacterium ◽  
Multiple Roles ◽  
Lysine Biosynthesis ◽  
Lag Phase ◽  
Gene Encoding ◽  
Branched Chain Amino Acid ◽  
The Tca Cycle ◽  
Branched Chain

The extremely thermophilic bacterium Thermus thermophilus HB27 synthesizes lysine through α-aminoadipate (AAA). In this study, a T. thermophilus gene encoding the enzyme that catalyses transamination of AAA was cloned as a mammalian kynurenine/AAA aminotransferase (Kat2) gene homologue. A T. thermophilus mutant with disruption of the Kat2 homologue required a longer lag phase for growth and showed slower growth in minimal medium. Furthermore, addition of AAA or lysine shortened the lag phase and improved the growth rate. The Kat2 homologue was therefore termed lysN. LysN recognizes not only 2-oxoadipate, an intermediate of lysine biosynthesis, but also 2-oxoisocaproate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, intermediates of leucine, valine and isoleucine biosyntheses, respectively, along with oxaloacetate, a compound in the TCA cycle, as an amino acceptor. These results suggest multiple roles of LysN in several cellular metabolic pathways including lysine and branched-chain amino acid biosyntheses.

scholarly journals Novel insights into development of diabetic bladder disorder provided by metabolomic analysis of the rat nondiabetic and diabetic detrusor and urothelial layer

2016 ◽  
Vol 311 (2) ◽  
pp. E471-E479 ◽  
Author(s):  
Yi Wang ◽  
Gary G. Deng ◽  
Kelvin P. Davies
Keyword(s):  
Degradation Products ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Branched Chain Amino Acid ◽  
The Tca Cycle ◽  
Glycation End Products ◽  
Markers Of Oxidative Stress ◽  
Branched Chain ◽  
Diabetic Bladder ◽  
Bladder Disorder

There are at present no published studies providing a global overview of changes in bladder metabolism resulting from diabetes. Such studies have the potential to provide mechanistic insight into the development of diabetic bladder disorder (DBD). In the present study, we compared the metabolome of detrusor and urothelial layer in a 1-mo streptozotocin-induced rat model of type 1 diabetes with nondiabetic controls. Our studies revealed that diabetes caused both common and differential changes in the detrusor and urothelial layer's metabolome. Diabetes resulted in similar changes in the levels of previously described diabetic markers in both tissues, such as glucose, lactate, 2-hydroxybutyrate, branched-chain amino acid degradation products, bile acids, and 1,5-anhydroglucitol, as well as markers of oxidative stress. In the detrusor (but not the urothelial layer), diabetes caused activation of the pentose-phosphate and polyol pathways, concomitant with a reduction in the TCA cycle and β-oxidation. Changes in detrusor energy-generating pathways resulted in an accumulation of sorbitol that, through generation of advanced glycation end products, is likely to play a central role in the development of DBD. In the diabetic urothelial layer there was decreased flux of glucose via glycolysis and changes in lipid metabolism, particularly prostaglandin synthesis, which also potentially contributes to detrusor dysfunction.

scholarly journals Ammonia inhibits energy metabolism in astrocytes in a rapid and glutamate dehydrogenase 2-dependent manner

2020 ◽  
Vol 13 (10) ◽  
pp. dmm047134
Author(s):  
Leonie Drews ◽  
Marcel Zimmermann ◽  
Philipp Westhoff ◽  
Dominik Brilhaus ◽  
Rebecca E. Poss ◽  
...  
Keyword(s):  
Amino Acids ◽  
Energy Metabolism ◽  
Glutamate Dehydrogenase ◽  
Mitochondrial Respiration ◽  
Tca Cycle ◽  
Dependent Manner ◽  
Independent Manner ◽  
The Tca Cycle ◽  
Branched Chain ◽  
Tca Cycle Intermediates

ABSTRACTAstrocyte dysfunction is a primary factor in hepatic encephalopathy (HE) impairing neuronal activity under hyperammonemia. In particular, the early events causing ammonia-induced toxicity to astrocytes are not well understood. Using established cellular HE models, we show that mitochondria rapidly undergo fragmentation in a reversible manner upon hyperammonemia. Further, in our analyses, within a timescale of minutes, mitochondrial respiration and glycolysis were hampered, which occurred in a pH-independent manner. Using metabolomics, an accumulation of glucose and numerous amino acids, including branched chain amino acids, was observed. Metabolomic tracking of 15N-labeled ammonia showed rapid incorporation of 15N into glutamate and glutamate-derived amino acids. Downregulating human GLUD2 [encoding mitochondrial glutamate dehydrogenase 2 (GDH2)], inhibiting GDH2 activity by SIRT4 overexpression, and supplementing cells with glutamate or glutamine alleviated ammonia-induced inhibition of mitochondrial respiration. Metabolomic tracking of 13C-glutamine showed that hyperammonemia can inhibit anaplerosis of tricarboxylic acid (TCA) cycle intermediates. Contrary to its classical anaplerotic role, we show that, under hyperammonemia, GDH2 catalyzes the removal of ammonia by reductive amination of α-ketoglutarate, which efficiently and rapidly inhibits the TCA cycle. Overall, we propose a critical GDH2-dependent mechanism in HE models that helps to remove ammonia, but also impairs energy metabolism in mitochondria rapidly.

scholarly journals BCAT1 affects mitochondrial metabolism independently of leucine transamination in activated human macrophages

2020 ◽  
Vol 133 (22) ◽  
pp. jcs247957
Author(s):  
Jeong-Hun Ko ◽  
Antoni Olona ◽  
Adonia E. Papathanassiu ◽  
Norzawani Buang ◽  
Kwon-Sik Park ◽  
...  
Keyword(s):  
Macrophage Activation ◽  
Polarization State ◽  
Tca Cycle ◽  
Metabolic Adaptation ◽  
Human Macrophages ◽  
Relative Contribution ◽  
The Tca Cycle ◽  
Nutrient Consumption ◽  
Anti Oxidant ◽  
Branched Chain

ABSTRACTIn response to environmental stimuli, macrophages change their nutrient consumption and undergo an early metabolic adaptation that progressively shapes their polarization state. During the transient, early phase of pro-inflammatory macrophage activation, an increase in tricarboxylic acid (TCA) cycle activity has been reported, but the relative contribution of branched-chain amino acid (BCAA) leucine remains to be determined. Here, we show that glucose but not glutamine is a major contributor of the increase in TCA cycle metabolites during early macrophage activation in humans. We then show that, although uptake of BCAAs is not altered, their transamination by BCAT1 is increased following 8 h lipopolysaccharide (LPS) stimulation. Of note, leucine is not metabolized to integrate into the TCA cycle in basal or stimulated human macrophages. Surprisingly, the pharmacological inhibition of BCAT1 reduced glucose-derived itaconate, α-ketoglutarate and 2-hydroxyglutarate levels without affecting succinate and citrate levels, indicating a partial inhibition of the TCA cycle. This indirect effect is associated with NRF2 (also known as NFE2L2) activation and anti-oxidant responses. These results suggest a moonlighting role of BCAT1 through redox-mediated control of mitochondrial function during early macrophage activation.

scholarly journals Regulation of the acuF Gene, Encoding Phosphoenolpyruvate Carboxykinase in the Filamentous Fungus Aspergillus nidulans

2002 ◽  
Vol 184 (1) ◽  
pp. 183-190 ◽  
Author(s):  
Michael J. Hynes ◽  
Oliver W. Draht ◽  
Meryl A. Davis
Keyword(s):  
Carbon Source ◽  
Aspergillus Nidulans ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Carbon Sources ◽  
Tca Cycle ◽  
Northern Blotting ◽  
Gene Encoding ◽  
The Tca Cycle ◽  
Key Enzyme ◽  
Acetate Utilization

ABSTRACT Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme required for gluconeogenesis when microorganisms grow on carbon sources metabolized via the tricarboxylic acid (TCA) cycle. Aspergillus nidulans acuF mutants isolated by their inability to use acetate as a carbon source specifically lack PEPCK. The acuF gene has been cloned and shown to encode a protein with high similarity to PEPCK from bacteria, plants, and fungi. The regulation of acuF expression has been studied by Northern blotting and by the construction of lacZ fusion reporters. Induction by acetate is abolished in mutants unable to metabolize acetate via the TCA cycle, and induction by amino acids metabolized via 2-oxoglutarate is lost in mutants unable to form 2-oxoglutarate. Induction by acetate and proline is not additive, consistent with a single mechanism of induction. Malate and succinate result in induction, and it is proposed that PEPCK is controlled by a novel mechanism of induction by a TCA cycle intermediate or derivative, thereby allowing gluconeogenesis to occur during growth on any carbon source metabolized via the TCA cycle. It has been shown that the facB gene, which mediates acetate induction of enzymes specifically required for acetate utilization, is not directly involved in PEPCK induction. This is in contrast to Saccharomyces cerevisiae, where Cat8p and Sip4p, homologs of FacB, regulate PEPCK as well as the expression of other genes necessary for growth on nonfermentable carbon sources in response to the carbon source present. This difference in the control of gluconeogenesis reflects the ability of A. nidulans and other filamentous fungi to use a wide variety of carbon sources in comparison with S. cerevisiae. The acuF gene was also found to be subject to activation by the CCAAT binding protein AnCF, a protein homologous to the S. cerevisiae Hap complex and the mammalian NFY complex.

scholarly journals Branched-chain amino acid catabolism depends on GRXS15 through mitochondrial lipoyl cofactor homeostasis

2020 ◽  
Author(s):  
Anna Moseler ◽  
Inga Kruse ◽  
Andrew E. Maclean ◽  
Luca Pedroletti ◽  
Stephan Wagner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Degradation Products ◽  
Tca Cycle ◽  
Cluster Assembly ◽  
Amino Acid Catabolism ◽  
Metabolic Defects ◽  
Branched Chain Amino Acid ◽  
Branched Chain ◽  
Dependent Processes

AbstractIron-sulfur (Fe-S) clusters are ubiquitous cofactors in all life and are used in a wide array of diverse biological processes, including electron transfer chains and several metabolic pathways. Biosynthesis machineries for Fe-S clusters exist in plastids, the cytosol and mitochondria. A single monothiol glutaredoxin (GRX) has been shown to be involved in Fe-S cluster assembly in mitochondria of yeast and mammals. In plants, the role of the mitochondrial homologue GRXS15 has only partially been characterized. Arabidopsis grxs15 null mutants are not viable, but mutants complemented with the variant GRXS15 K83A develop with a dwarf phenotype. In an in-depth metabolic analysis, we show that most Fe-S cluster-dependent processes are not affected, including biotin biosynthesis, molybdenum cofactor biosynthesis and the electron transport chain. Instead, we observed an increase in most TCA cycle intermediates and amino acids, especially pyruvate, 2-oxoglutarate, glycine and branched-chain amino acids (BCAAs). The most pronounced accumulation occurred in branched-chain α-keto acids (BCKAs), the first degradation products resulting from deamination of BCAAs. In wild-type plants, pyruvate, 2-oxoglutarate, glycine and BCKAs are all metabolized through decarboxylation by four mitochondrial lipoyl cofactor-dependent dehydrogenase complexes. Because these enzyme complexes are very abundant and the biosynthesis of the lipoyl cofactor depends on continuous Fe-S cluster supply to lipoyl synthase, this could explain why lipoyl cofactor-dependent processes are most sensitive to restricted Fe-S supply in GRXS15 K83A mutants.One-sentence summaryDeficiency in GRXS15 restricts protein lipoylation and causes metabolic defects in lipoyl cofactor-dependent dehydrogenase complexes, with branched-chain amino acid catabolism as dominant bottleneck.

scholarly journals Physiological Biochemistry-Combined Transcriptomic Analysis Reveals Mechanism of Bacillus cereus G2 Improved Salt-Stress Tolerance of Glycyrrhiza uralensis Fisch. Seedlings by Balancing Carbohydrate Metabolism

2022 ◽  
Vol 12 ◽  
Author(s):  
Xiang Xiao ◽  
Qiuli Wang ◽  
Xin Ma ◽  
Duoyong Lang ◽  
Zhenggang Guo ◽  
...  
Keyword(s):  
Salt Stress ◽  
Bacillus Cereus ◽  
Starch Content ◽  
Tca Cycle ◽  
Soluble Starch ◽  
Glycyrrhiza Uralensis ◽  
Salt Stress Tolerance ◽  
Gene Encoding ◽  
The Tca Cycle ◽  
Carbohydrate Contents

Salt stress severely threatens the growth and productivity of Glycyrrhiza uralensis. Previous results found that Bacillus cereus G2 enhanced several carbohydrate contents in G. uralensis under salt stress. Here, we analyzed the changes in parameters related to growth, photosynthesis, carbohydrate transformation, and the glycolysis Embden-Meyerhof-Parnas (EMP) pathway-tricarboxylic acid (TCA) cycle by G2 in G. uralensis under salt stress. Results showed that G2 helped G. uralensis-accumulating photosynthetic pigments during photosynthesis, which could further increase starch, sucrose, and fructose contents during carbohydrate transformation. Specifically, increased soluble starch synthase (SSS) activity caused to higher starch content, which could induce α-amylase (AM) and β-amylase (BM) activities; increased sucrose content due to the increase of sucrose synthase (SS) activity through upregulating the gene-encoding SS, which decreased cell osmotic potential, and consequently, induced invertase and gene-encoding α-glucosidase that decomposed sucrose to fructose, ultimately avoided further water loss; increased fructose content-required highly hexokinase (HK) activity to phosphorylate in G. uralensis, thereby providing sufficient substrate for EMP. However, G2 decreased phosphofructokinase (PFK) and pyruvate kinase (PK) activities during EMP. For inducing the TCA cycle to produce more energy, G2 increased PDH activity that enhanced CA content, which further increased isocitrate dehydrogenase (ICDH) activity and provided intermediate products for the G. uralensis TCA cycle under salt stress. In sum, G2 could improve photosynthetic efficiency and carbohydrate transformation to enhance carbohydrate products, thereby releasing more chemical energy stored in carbohydrates through the EMP pathway-TCA cycle, finally maintain normal life activities, and promote the growth of G. uralensis under salt stress.

Cloning and characterization of brnQ, a gene encoding a low-affinity, branched-chain amino acid carrier in Lactobacillus delbrdückii subsp. lactic DSM7290

1995 ◽  
Vol 249 (6) ◽  
pp. 682-690 ◽  
Author(s):  
Klaus Stucky ◽  
Anja Hagting ◽  
Jargen R. Klein ◽  
Hugo Matern ◽  
Bernhard Henrich ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gene Encoding ◽  
Branched Chain Amino Acid ◽  
Branched Chain ◽  
Amino Acid Carrier

scholarly journals Branched-chain-amino-acid biosynthesis in plants: molecular cloning and characterization of the gene encoding acetohydroxy acid isomeroreductase (ketol-acid reductoisomerase) from Arabidopsis thaliana (thale cress)

1993 ◽  
Vol 294 (3) ◽  
pp. 821-828 ◽  
Author(s):  
R Dumas ◽  
G Curien ◽  
R T DeRose ◽  
R Douce
Keyword(s):  
Arabidopsis Thaliana ◽  
Amino Acid ◽  
Molecular Mechanisms ◽  
Amino Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
Gene Encoding ◽  
Translational Initiation ◽  
Branched Chain Amino Acid ◽  
Thale Cress ◽  
Branched Chain

Towards the goal of gaining a better understanding of the molecular mechanisms controlling branched-chain-amino-acid biosynthesis in plants, we have isolated, sequenced and characterized a gene encoding acetohydroxy acid isomero-reductase (ketol-acid reductoisomerase) from Arabidopsis thaliana (thale cress). Comparison between the acetohydroxy acid isomeroreductase cDNA and the genomic sequence has allowed us to determine the exon structure of the coding region. The isolated acetohydroxy acid isomeroreductase gene is distributed over approx. 4.5 kbp and contains nine introns (79-347 bp). The transcriptional start site was found to be 52 bp upstream of the translational initiation site. Southern-blot analysis of A. thaliana genomic DNA shows that the acetohydroxy acid isomeroreductase is encoded by a single-copy gene.

scholarly journals Toward an Understanding of the Structural and Mechanistic Aspects of Protein-Protein Interactions in 2-Oxoacid Dehydrogenase Complexes

Life ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 407
Author(s):  
Natalia S. Nemeria ◽  
Xu Zhang ◽  
Joao Leandro ◽  
Jieyu Zhou ◽  
Luying Yang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Tca Cycle ◽  
Degradation Pathway ◽  
Protein Protein Interactions ◽  
Gene Encoding ◽  
The Tca Cycle ◽  
Hybrid Complex ◽  
Key Enzyme ◽  
Lysine Degradation ◽  
Dehydrogenase Complex

The 2-oxoglutarate dehydrogenase complex (OGDHc) is a key enzyme in the tricarboxylic acid (TCA) cycle and represents one of the major regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. The OGDHc impacts cell metabolic and cell signaling pathways through the coupling of 2-oxoglutarate metabolism to gene transcription related to tumor cell proliferation and aging. DHTKD1 is a gene encoding 2-oxoadipate dehydrogenase (E1a), which functions in the L-lysine degradation pathway. The potentially damaging variants in DHTKD1 have been associated to the (neuro) pathogenesis of several diseases. Evidence was obtained for the formation of a hybrid complex between the OGDHc and E1a, suggesting a potential cross talk between the two metabolic pathways and raising fundamental questions about their assembly. Here we reviewed the recent findings and advances in understanding of protein-protein interactions in OGDHc and 2-oxoadipate dehydrogenase complex (OADHc), an understanding that will create a scaffold to help design approaches to mitigate the effects of diseases associated with dysfunction of the TCA cycle or lysine degradation. A combination of biochemical, biophysical and structural approaches such as chemical cross-linking MS and cryo-EM appears particularly promising to provide vital information for the assembly of 2-oxoacid dehydrogenase complexes, their function and regulation.

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