Synthesis of α,β-Deuterated 15N Amino Acids Using a Coupled Glutamate Dehydrogenase-Branched-Chain Amino Acid Aminotransferase System

1993 ◽  
Vol 213 (1) ◽  
pp. 147-151 ◽  
Author(s):  
J.A. Chanatry ◽  
P.H. Schafer ◽  
M.S. Kim ◽  
D.M. Lemaster
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glutamate Dehydrogenase ◽  
Branched Chain Amino Acid ◽  
Branched Chain

scholarly journals Rhizobium leguminosarum Has a Second General Amino Acid Permease with Unusually Broad Substrate Specificity and High Similarity to Branched-Chain Amino Acid Transporters (Bra/LIV) of the ABC Family

2002 ◽  
Vol 184 (15) ◽  
pp. 4071-4080 ◽  
Author(s):  
A. H. F. Hosie ◽  
D. Allaway ◽  
C. S. Galloway ◽  
H. A. Dunsby ◽  
P. S. Poole
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rhizobium Leguminosarum ◽  
Binding Protein ◽  
Amino Acid Transporters ◽  
Acid Uptake ◽  
Amino Acid Permease ◽  
Branched Chain Amino Acid ◽  
General Amino Acid Permease ◽  
Branched Chain

ABSTRACT Amino acid uptake by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (BraRl). Characterization of the solute specificity of BraRl shows it to be the second general amino acid permease of R. leguminosarum. Although BraRl has high sequence identity to members of the family of hydrophobic amino acid transporters (HAAT), it transports a broad range of solutes, including acidic and basic polar amino acids (l-glutamate, l-arginine, and l-histidine), in addition to neutral amino acids (l-alanine and l-leucine). While amino and carboxyl groups are required for transport, solutes do not have to be α-amino acids. Consistent with this, BraRl is the first ABC transporter to be shown to transport γ-aminobutyric acid (GABA). All previously identified bacterial GABA transporters are secondary carriers of the amino acid-polyamine-organocation (APC) superfamily. Also, transport by BraRl does not appear to be stereospecific as d amino acids cause significant inhibition of uptake of l-glutamate and l-leucine. Unlike all other solutes tested, l-alanine uptake is not dependent on solute binding protein BraCRl. Therefore, a second, unidentified solute binding protein may interact with the BraDEFGRl membrane complex during l-alanine uptake. Overall, the data indicate that BraRl is a general amino acid permease of the HAAT family. Furthermore, BraRl has the broadest solute specificity of any characterized bacterial amino acid transporter.

scholarly journals Dietary casein, egg albumin, and branched-chain amino acids attenuate phosphate-induced renal tubulointerstitial injury in rats

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Karin Shimada ◽  
Isao Matsui ◽  
Kazunori Inoue ◽  
Ayumi Matsumoto ◽  
Seiichi Yasuda ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Muscle Weakness ◽  
Branched Chain Amino Acids ◽  
Protective Effects ◽  
Egg Albumin ◽  
Branched Chain Amino Acid ◽  
Renal Tubulointerstitial Fibrosis ◽  
Dietary Casein ◽  
Branched Chain

Abstract Dietary phosphate intake is closely correlated with protein intake. However, the effects of the latter on phosphate-induced organ injuries remain uncertain. Herein, we investigated the effects of low (10.8%), moderate (23.0%), and high (35.2%) dietary casein and egg albumin administration on phosphate-induced organ injuries in rats. The moderate and high casein levels suppressed renal tubulointerstitial fibrosis and maintained mitochondrial integrity in the kidney. The serum creatinine levels were suppressed only in the high casein group. Phosphate-induced muscle weakness was also ameliorated by high dietary casein. The urinary and fecal phosphate levels in the early experiment stage showed that dietary casein did not affect phosphate absorption from the intestine. High dietary egg albumin showed similar kidney protective effects, while the egg albumin effects on muscle weakness were only marginally significant. As the plasma branched-chain amino acid levels were elevated in casein- and egg albumin-fed rats, we analyzed their effects. Dietary supplementation of 10% branched-chain amino acids suppressed phosphate-induced kidney injury and muscle weakness. Although dietary protein restriction is recommended in cases of chronic kidney disease, our findings indicate that the dietary casein, egg albumin, and branched-chain amino acid effects might be reconsidered in the era of a phosphate-enriched diet.

scholarly journals Mechanism of De Novo Branched-Chain Amino Acid Synthesis as an Alternative Electron Sink in Hypoxic Aspergillus nidulans Cells

2010 ◽  
Vol 76 (5) ◽  
pp. 1507-1515 ◽  
Author(s):  
Motoyuki Shimizu ◽  
Tatsuya Fujii ◽  
Shunsuke Masuo ◽  
Naoki Takaya
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aspergillus Nidulans ◽  
De Novo ◽  
Acid Synthesis ◽  
Branched Chain Amino Acids ◽  
Amino Acid Synthesis ◽  
Lactate Dehydrogenase Activity ◽  
Branched Chain Amino Acid ◽  
Branched Chain

ABSTRACT Although branched-chain amino acids are synthesized as building blocks of proteins, we found that the fungus Aspergillus nidulans excretes them into the culture medium under hypoxia. The transcription of predicted genes for synthesizing branched-chain amino acids was upregulated by hypoxia. A knockout strain of the gene encoding the large subunit of acetohydroxy acid synthase (AHAS), which catalyzes the initial reaction of the synthesis, required branched-chain amino acids for growth and excreted very little of them. Pyruvate, a substrate for AHAS, increased the amount of hypoxic excretion in the wild-type strain. These results indicated that the fungus responds to hypoxia by synthesizing branched-chain amino acids via a de novo mechanism. We also found that the small subunit of AHAS regulated hypoxic branched-chain amino acid production as well as cellular AHAS activity. The AHAS knockout resulted in higher ratios of NADH/NAD+ and NADPH/NADP+ under hypoxia, indicating that the branched-chain amino acid synthesis contributed to NAD+ and NADP+ regeneration. The production of branched-chain amino acids and the hypoxic induction of involved genes were partly repressed in the presence of glucose, where cells produced ethanol and lactate and increased levels of lactate dehydrogenase activity. These indicated that hypoxic branched-chain amino acid synthesis is a unique alternative mechanism that functions in the absence of glucose-to-ethanol/lactate fermentation and oxygen respiration.

scholarly journals Biochemical and structural characterization of a highly active branched-chain amino acid aminotransferase from Pseudomonas sp. for efficient biosynthesis of chiral amino acids

2019 ◽  
Author(s):  
Xinxin Zheng ◽  
Yinglu Cui ◽  
Tao Li ◽  
Ruifeng Li ◽  
Lu Guo ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Catalytic Mechanism ◽  
Chiral Amines ◽  
Branched Chain Amino Acid ◽  
Chiral Amino Acids ◽  
Branched Chain ◽  
Substrate Spectrum

AbstractAminotransferases (ATs) are important biocatalysts for the synthesis of chiral amines because of their capability of introducing amino group into ketones or keto acids as well as their high enantioselectivity, high regioselectivity and no requirement of external addition of cofactor. Among all ATs, branched-chain amino acid aminotransferase (BCAT) can reversibly catalyse branched-chain amino acids (BCAAs), including L-valine, L-leucine, and L-isoleucine, with α-ketoglutaric acid to form the corresponding ketonic acids and L-glutamic acid. Alternatively, BCATs have been used for the biosynthesis of unnatural amino acids, such as L-tert-leucine. In the present study, the BCAT from Pseudomonas sp. (PsBCAT) was cloned and expressed in Escherichia coli for biochemical and structural analyses. The optimal reaction temperature and pH of PsBCAT were 40 °C and 8.5, respectively. PsBCAT exhibited a comparatively broader substrate spectrum, and showed remarkably high activity with L-leucine, L-valine, L-isoleucine and L-methionine with activities of 105 U/mg, 127 U/mg, 115 U/mg and 98 U/mg, respectively. Additionally, PsBCAT had activities with aromatic L-amino acids, L-histidine, L-lysine, and L-threonine. To analyse the catalytic mechanism of PsBCAT with the broad substrate spectrum, the crystal structure of PsBCAT was also determined. Finally, conjugated with the ornithine aminotransferase (OrnAT) from Bacillus subtilis, the coupled system was applied to the preparation of L-tert-leucine with 83% conversion, which provided an approximately 2.7-fold higher yield than the single BCAT reaction.IMPORTANCEDespite the enormous potential of BCATs, the vast majority of enzymes still lack suitably broad substrate scope and activity, thus new sources and novel enzymes are currently being investigated. Here, we described a previously uncharacterized PsBCAT, which showed a surprisingly wide substrate range and was more active towards BCAAs. This substrate promiscuity is unique for the BCAT family and could prove useful in industrial applications. Based on the determined crystal structure, we found some differences in the organization of the substrate binding cavity, which may influence the substrate specificity of the enzyme. Moreover, we demonstrated efficient biocatalytic asymmetric synthesis of L-tert-leucine using a coupling system, which can be used to remove the inhibitory by-product, and to shift the reaction equilibrium towards the product formation. In summary, the structural and functional characteristics of PsBCAT were analysed in detail, and this information will play an important role in the synthesis of chiral amino acids and will be conducive to industrial production of enantiopure chiral amines by aminotransferase.

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 Quantitative Analysis of Branched-chain Amino Acids in Five Nutritional Supplements Using a Leucine Dehydrogenase Assay (P23-004-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Brendan Eley
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Nutritional Supplements ◽  
Sport Nutrition ◽  
Free Form ◽  
Leucine Dehydrogenase ◽  
Funding Sources ◽  
Branched Chain Amino Acid ◽  
Branched Chain ◽  
The Given

Abstract Objectives The objective of this experiment was to determine the branched-chain amino acid (BCAA) content in five different sport nutrition supplements compared to the amount claimed on the label. Methods To measure the BCAA content of five nutritional supplements, a leucine dehydrogenase enzyme assay was used. This enzyme catalyzes the reaction of turning the given BCAA (L-leucine, L-valine, and L-isoleucine), water, and NAD+ into their respective metabolite, NH3, NADH, and H+. Ultraviolet-visible light spectrophotometry (UV/Vis) was used at 340 nm to create a standard curve. This curve uses the Beer-Lambert Law to measure NADH concentration from absorbance. NADH is in a 1:1 ratio with each BCAA molecule thus relaying the content of the given sample. The assay is specific to the three BCAAs in their free form. Other amino acids, as well as BCAAs in oligopeptides, do not interfere with this experiment. Products including oligo- and polypeptides were not included for testing. The assay was performed for each product and ran against a known standard (≥98% L-leucine) for validation. Due to different supplements having different BCAA amounts per serving, % content of the claimed amount was measured. Results Compared to the amount provided by the labels of each supplement, BCAA content was on average only 61% of the manufacturer claims when compared to ≥98% L-leucine. This shows that these BCAA supplements do not meet label claims for BCAA content (P < 0.01). Conclusions The five tested nutritional supplements contain significantly less branched-chain amino acid content than claimed on the label. This experiment can be expanded on in the future to test content of other BCAA containing supplements to determine how common underdosing is in the industry as a whole. Funding Sources The author claims no funding sources.

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