Some enzymic syntheses of 15N-L-aspartic acid and 15N-L-glutamic acid

1969 ◽  
Vol 47 (3) ◽  
pp. 411-415 ◽  
Author(s):  
J. A. Zintel ◽  
A. J. Williams ◽  
R. S. Stuart
Keyword(s):  
Glutamic Acid ◽  
Aspartic Acid ◽  
Fumaric Acid ◽  
Aspartate Aminotransferase ◽  
Efficient Utilization ◽  
E Coli ◽  
Acid Dehydrogenase ◽  
Acid Catalyzed ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

Some methods for the preparation of 15N-L-aspartic acid and 15N-L-glutamic acid using enzyme catalyzed reactions are described. 15N-L-Aspartic acid is prepared by the addition of ammonia to fumaric acid, catalyzing the reaction with aspartase which was partially purified from E. coli. 15N-L-Glutamic acid with a high 15N/14N ratio is prepared by transamination with 15N-L-aspartic acid catalyzed by aspartate aminotransferase. 15N-L-Glutamic acid with a low 15N/14N ratio is prepared by a glutamic acid dehydrogenase catalyzed exchange of L-glutamic acid with 15N-ammonia. These enzymes are commercially available. Since efficient utilization of 15N is obtained, aspartic acid or glutamic acid may be prepared with any desired 15N/14N ratio.

Stoffwechselregulation durch den Zellwandbaustein Kieselsäure: Polgrößenänderungen von a-Ketoglutarsäure, Aminosäuren und Nucleosidphosphaten

1968 ◽  
Vol 23 (2) ◽  
pp. 268-271 ◽  
Author(s):  
D. Werner
Keyword(s):  
Glutamic Acid ◽  
Aspartic Acid ◽  
Rna Synthesis ◽  
Acid Synthesis ◽  
Centric Diatom ◽  
Free Medium ◽  
Acetyl Coa ◽  
E Coli ◽  
Acid Pool ◽  
Acetyl Coa Pathway

When the centric diatom Cyclotella cryptica is grown in a Si (OH) 4-free medium, the glutamic acid pool decreases within 3 hours to a third of the original value, whereas the aspartic acid pool is reduced by only about 20 per cent. The pools of nucleosid-triphosphates and of glycerol-1-phosphate remain unaffected during this time. The nucleosid-diphosphates pool decreases in the same way as that of aspartic acid. The decrease in the glutamic acid pool precedes the inhibition of total protein synthesis in Si (OH) 4-deficient cells, and a significant decrease in the a-ketoglutarate pool precedes the decrease of the glutamic acid content. Already within 60 minutes ofter incubation in a Si (OH) 4-free medium, the content of a-ketoglutarate is decreased to one third of the normal value. On the other hand, the acetyl CoA pathway (enhanced fatty acid synthesis) is not inhibited. The results suggest, that the Si (OH) 4-metabolism interferes with reactions between the condensing enzyme (acetyl-CoA and oxalacetate) and a-ketoglutarate. The delay between inhibition of protein- and RNA-synthesis and the different changes in the pools of amino acids and nucleosid-triphosphates resemble the regulation of the nucleosid-triphosphate pool and RNA-synthesis in amino acid starved strains of E. coli (EDLIN and NEUHARD) 1, though the primary causes are quite different.

Regulation of Glyconeogenesis from Amino Acids by Acetate in Tetrahymena pyriformis

1973 ◽  
Vol 51 (4) ◽  
pp. 323-331 ◽  
Author(s):  
C. Mavrides
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Glutamate Dehydrogenase ◽  
Aspartic Acid ◽  
Growth Medium ◽  
Aspartate Aminotransferase ◽  
Alanine Aminotransferase ◽  
Sodium Acetate ◽  
Tetrahymena Pyriformis

The regulation of glyconeogenesis from amino acids by acetate was studied in Tetrahymena pyriformis. Alanine aminotransferase and glutamate dehydrogenase were repressed by 0.1% sodium acetate in the growth medium. Incorporations into the glycogen of washed cells from the respective isotopically labelled amino acids were similarly suppressed.Incorporations into glycogen from uniformly 14C-labelled L-serine, L-leucine, L-isoleucine, L-tyrosine, and DL-β-14C-tyrosine were also suppressed by prior growth in a medium supplemented with 0.1% or 0.3% acetate. Percentage incorporation into glycogen was highest from tyrosine, followed by leucine, isoleucine, and alanine, and lowest from glutamic acid and serine.Supplementation of the medium with 0.25% glucose resulted in repression of the above two enzymes and suppression of incorporation into glycogen from amino acids.Incorporation of aspartic acid into glycogen was negligible and was variously and minimally affected by growth in glucose- or acetate-supplemented media. Aspartate aminotransferase was affected in a like manner.Glycogen content was not significantly affected by growth in media supplemented with 0.1% or 0.3% acetate. On the whole, the data suggest that acetate spares amino acids for glyconeogenesis by a mechanism which entails repression of amino-acid-catabolizing enzymes.

scholarly journals An In Vivo Biocatalytic Cascade Featuring an Artificial Enzyme Catalyzed New-to-Nature Reaction

2021 ◽  
Author(s):  
Linda Ofori Atta ◽  
Zhi Zhou ◽  
Gerard Roelfes
Keyword(s):  
Carbonyl Compounds ◽  
Artificial Enzymes ◽  
Catalytic Residue ◽  
Artificial Enzyme ◽  
E Coli ◽  
Iminium Ion ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Benzaldehyde Derivatives

Artificial enzymes utilizing the genetically encoded non-proteinogenic amino acid p-aminophenylalanine (pAF) as catalytic residue are able to react with carbonyl compounds through an iminium ion mechanism, making reactions possible that have no equivalent in nature. Here, we report an in vivo biocatalytic cascade that is augmented with such an artificial enzyme catalyzed new-to-nature reaction. The artificial enzyme in this study is a pAF containing evolved variant of the Lactococcal multidrug resistance Regulator, designated LmrR_V15pAF_RMH, which efficiently converts in vivo produced benzaldehyde derivatives into the corresponding hydrazone products inside E. coli cells. These in vivo biocatalytic cascades comprising an artificial enzyme catalyzed reactions are an important step towards achieving a hybrid metabolism.

scholarly journals Development of a vanillate biosensor for the vanillin biosynthesis pathway in E. coli

2018 ◽  
Author(s):  
Aditya M. Kunjapur ◽  
Kristala L. J. Prather
Keyword(s):  
Metabolic Engineering ◽  
Dynamic Range ◽  
De Novo ◽  
Caulobacter Crescentus ◽  
Homo Sapiens ◽  
Biosynthesis Pathway ◽  
Dynamic Regulation ◽  
E Coli ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

AbstractGenetically encoded small molecule sensors can facilitate metabolic engineering by enabling high-throughput detection of metabolite concentrations, directed evolution of host and pathway enzymes, and dynamic regulation. The engineered de novo vanillin biosynthesis pathway assembled in Escherichia coli is industrially relevant and ideal for biosensor deployment given that the pathway requires only three heterologous enzyme-catalyzed reactions, generates naturally occurring metabolites, and may benefit from dynamic regulation. However, pathway flux is stalled and diverted by the activity of the Homo sapiens catechol O-methyltransferase, which is intended to catalyze the conversion of protocatechuate to vanillate. To confront this challenge, we constructed and applied a vanillate sensor based on the Caulobacter crescentus VanR-VanO system. Using components from a previously characterized E. coli promoter library, we achieved greater than 14-fold dynamic range in our best rationally constructed sensor. We characterized sensor substrate specificity and found that this construct and an evolved variant are remarkably selective, exhibiting no detectable response to the regioisomer byproduct isovanillate. We then harnessed the evolved biosensor to conduct rapid bioprospecting of natural catechol O-methyltransferases. We identified eight that appear to have greater desired activity than the originally used variant, including three previously uncharacterized O-methyltransferases. Collectively, these efforts enrich our knowledge of how biosensing can aid metabolic engineering and constitute the foundation for future improvements in vanillin pathway productivity.

A kinetic analysis of coupled sequential enzyme reactions

2018 ◽  
Author(s):  
Justin Eilertsen ◽  
Santiago Schnell
Keyword(s):  
Enzyme Assay ◽  
Sequential Reaction ◽  
Enzyme Reactions ◽  
Indicator Reaction ◽  
Single Substrate ◽  
Complex Sequences ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Single Enzyme

<div>As a case study, we consider a coupled enzyme assay of sequential enzyme reactions obeying the Michaelis--Menten reaction mechanism. The sequential reaction consists of a single-substrate, single-enzyme non-observable reaction followed by another single-substrate, single-enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction becomes the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the sequential reaction is described by a pair of interacting Michaelis--Menten equations. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis--Menten equations are derived. The theory can be extended to deal with more complex sequences of enzyme catalyzed reactions.</div>

A kinetic analysis of coupled sequential enzyme reactions

2018 ◽  
Author(s):  
Justin Eilertsen ◽  
Santiago Schnell
Keyword(s):  
Enzyme Assay ◽  
Sequential Reaction ◽  
Enzyme Reactions ◽  
Indicator Reaction ◽  
Single Substrate ◽  
Complex Sequences ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Single Enzyme

<div>As a case study, we consider a coupled enzyme assay of sequential enzyme reactions obeying the Michaelis-Menten reaction mechanism. The sequential reaction consists of a single-substrate, single enzyme non-observable reaction followed by another single-substrate, single enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction becomes the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the sequential reaction is described by a pair of interacting Michaelis-Menten equations. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis-Menten equations are derived. The theory can be extended to deal with more complex sequences of enzyme catalyzed reactions.</div>

Computationally Augmented Retrosynthesis: Total Synthesis of Paspaline A and Emindole PB

2018 ◽  
Author(s):  
Timothy Newhouse ◽  
Daria E. Kim ◽  
Joshua E. Zweig
Keyword(s):  
Chemical Synthesis ◽  
Total Synthesis ◽  
Small Molecules ◽  
First Principles ◽  
Quantum Chemical ◽  
Computational Analysis ◽  
Empirical Evaluation ◽  
Synthetic Strategy ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

The diverse molecular architectures of terpene natural products are assembled by exquisite enzyme-catalyzed reactions. Successful recapitulation of these transformations using chemical synthesis is hard to predict from first principles and therefore challenging to execute. A means of evaluating the feasibility of such chemical reactions would greatly enable the development of concise syntheses of complex small molecules. Herein, we report the computational analysis of the energetic favorability of a key bio-inspired transformation, which we use to inform our synthetic strategy. This approach was applied to synthesize two constituents of the historically challenging indole diterpenoid class, resulting in a concise route to (–)-paspaline A in 9 steps from commercially available materials and the first pathway to and structural confirmation of emindole PB in 13 steps. This work highlights how traditional retrosynthetic design can be augmented with quantum chemical calculations to reveal energetically feasible synthetic disconnections, minimizing time-consuming and expensive empirical evaluation.

Photoinduced rearrangement of hydroxymethylisoxazolines, a route to enaminoaldehydes

1986 ◽  
Vol 51 (10) ◽  
pp. 2167-2180 ◽  
Author(s):  
Lubor Fišera ◽  
Nadezhda D. Kozhina ◽  
Peter Oravec ◽  
Hans-Joachim Timpe ◽  
Ladislav Štibrányi ◽  
...  
Keyword(s):  
Thionyl Chloride ◽  
Quantum Yields ◽  
Acid Catalyzed ◽  
Catalyzed Reactions ◽  
Photoinduced Rearrangement ◽  
Condensed Heterocycles

3-Aryl-4-R-carbamoyl-5-hydroxymethylisoxazolines (IV) were synthesized by allowing R-NH2 amines with R = H, CH3, C3H7, C6H5C2H5, and NH2 to act on 3-(X-phenyl)-4-oxo-3a,4,6,6a-tetrahydrofuro[3,4-d]isoxazoles (III) with X = H, 4-CH3, 4-OCH3, 2-OCH3, 4-Cl, 2-Cl, 4-F, 2-F, 4-Br, 4-NO2, and 3-NO2. Exposed to radiation, the substances IV give Z-2-hydroxymethylamino-2-aryl-1-formylacrylamides (V) in good yields. The 4-Cl and 4-F substituted Z-derivatives V isomerize irreversibly to the E-derivatives VI if allowed to stand in solvent; the remaining derivatives V are stable. The quantum yields of the photoreaction are from 0.012 to 0.106 in dependence on the substituent X. In all cases where the compounds IV were used for the preparation of condensed heterocycles in conditions of acid-catalyzed reactions, lactones III were preferentially formed; the action of thionyl chloride on IV results in the formation of chloromethyl derivatives VIII, which do not undergo further cyclization.

Effect of cations on the activity of L-glutamic acid dehydrogenase

Life Sciences ◽  
1963 ◽  
Vol 2 (11) ◽  
pp. 834-839 ◽  
Author(s):  
E. Schoffeniels ◽  
R. Gilles
Keyword(s):  
Glutamic Acid ◽  
Acid Dehydrogenase
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