An enzymatic activation of formaldehyde for nucleotide methylation

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

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Chemical Stability of Ascorbic Acid Integrated into Commercial Products: A Review on Bioactivity and Delivery Technology

Antioxidants ◽

10.3390/antiox11010153 ◽

2022 ◽

Vol 11 (1) ◽

pp. 153

Author(s):

Xin Yin ◽

Kaiwen Chen ◽

Hao Cheng ◽

Xing Chen ◽

Shuai Feng ◽

...

Keyword(s):

Ascorbic Acid ◽

Active Site ◽

De Novo ◽

Food Matrix ◽

De Novo Synthesis ◽

Nutritional Characteristics ◽

Gulonolactone Oxidase ◽

Key Enzyme ◽

Commercial Applications ◽

Delivery Technology

The L-enantiomer of ascorbic acid is commonly known as vitamin C. It is an indispensable nutrient and plays a key role in retaining the physiological process of humans and animals. L-gulonolactone oxidase, the key enzyme for the de novo synthesis of ascorbic acid, is lacking in some mammals including humans. The functionality of ascorbic acid has prompted the development of foods fortified with this vitamin. As a natural antioxidant, it is expected to protect the sensory and nutritional characteristics of the food. It is thus important to know the degradation of ascorbic acid in the food matrix and its interaction with coexisting components. The biggest challenge in the utilization of ascorbic acid is maintaining its stability and improving its delivery to the active site. The review also includes the current strategies for stabilizing ascorbic acid and the commercial applications of ascorbic acid.

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De Novo Synthesis of Amino Acids by the Ruminal Bacteria Prevotella bryantii B14,Selenomonas ruminantium HD4, and Streptococcus bovis ES1

Applied and Environmental Microbiology ◽

10.1128/aem.64.8.2836-2843.1998 ◽

1998 ◽

Vol 64 (8) ◽

pp. 2836-2843 ◽

Cited By ~ 34

Author(s):

Cengiz Atasoglu ◽

Carmen Valdés ◽

Nicola D. Walker ◽

C. James Newbold ◽

R. John Wallace

Keyword(s):

Amino Acids ◽

Amino Acid ◽

De Novo ◽

Dependent Manner ◽

Streptococcus Bovis ◽

De Novo Synthesis ◽

Ruminal Bacteria ◽

Total N ◽

Selenomonas Ruminantium ◽

Prevotella Bryantii

ABSTRACT The influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria, Prevotella bryantii B14, Selenomonas ruminantiumHD4, and Streptococcus bovis ES1, was determined by growing these bacteria in media containing 15NH4Cl and various additions of pancreatic hydrolysates of casein (peptides) or amino acids. The proportion of cell N and amino acids formed de novo decreased as the concentration of peptides increased. At high concentrations of peptides (10 and 30 g/liter), the incorporation of ammonia accounted for less than 0.16 of bacterial amino acid N and less than 0.30 of total N. At 1 g/liter, which is more similar to peptide concentrations found in the rumen, 0.68, 0.87, and 0.46 of bacterial amino acid N and 0.83, 0.89, and 0.64 of total N were derived from ammonia by P. bryantii, S. ruminantium, andS. bovis, respectively. Concentration-dependent responses were also obtained with amino acids. No individual amino acid was exhausted in any incubation medium. For cultures of P. bryantii, peptides were incorporated and stimulated growth more effectively than amino acids, while cultures of the other species showed no preference for peptides or amino acids. Apparent growth yields increased by between 8 and 57%, depending on the species, when 1 g of peptides or amino acids per liter was added to the medium. Proline synthesis was greatly decreased when peptides or amino acids were added to the medium, while glutamate and aspartate were enriched to a greater extent than other amino acids under all conditions. Thus, the proportion of bacterial protein formed de novo in noncellulolytic ruminal bacteria varies according to species and the form and identity of the amino acid and in a concentration-dependent manner.

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A de novo Synthesis of Oxindoles from Cyclohexanone-Derived γ-Keto-ester Acceptors Using a Desaturative Amination–Cyclization Approach

Synthesis ◽

10.1055/a-1538-8429 ◽

2021 ◽

Author(s):

Henry P. Caldora ◽

Sebastian Govaerts ◽

Shashikant U. Dighe ◽

Oliver J. Turner ◽

Daniele Leonori

Keyword(s):

Amino Acids ◽

Primary Amine ◽

De Novo ◽

Building Blocks ◽

De Novo Synthesis ◽

Keto Ester ◽

Mild Conditions ◽

Blockbuster Drug

Here we report a desaturative approach for oxindole synthesis. This method uses simple γ-ester-containing cyclohexanones and primary amine building blocks as coupling partners. A dual photoredox–cobalt manifold is used to generate a secondary aniline that, upon heating, cyclizes with the pendent ester functionality. The process operates under mild conditions and was applied to the modification of several amino acids, the blockbuster drug mexiletine, as well as the formation of dihydroquinolinones.

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Inhibiting Methanogenesis Stimulated de novo Synthesis of Microbial Amino Acids in Mixed Rumen Batch Cultures Growing on Starch but not on Cellulose

Microorganisms ◽

10.3390/microorganisms8060799 ◽

2020 ◽

Vol 8 (6) ◽

pp. 799

Author(s):

Emilio M. Ungerfeld ◽

M. Fernanda Aedo ◽

Camila Muñoz ◽

Natalie L. Urrutia ◽

Emilio D. Martínez ◽

...

Keyword(s):

Amino Acids ◽

De Novo ◽

Nitrogen Sources ◽

Environmental Benefits ◽

De Novo Synthesis ◽

Protein Nitrogen ◽

Protein Supplements ◽

Batch Cultures ◽

Sufficient Energy ◽

Ruminant Diets

Ameliorating methane (CH4) emissions from ruminants would have environmental benefits, but it is necessary to redirect metabolic hydrogen ([H]) toward useful sinks to also benefit animal productivity. We hypothesized that inhibiting rumen methanogenesis would increase de novo synthesis of microbial amino acids (AA) as an alternative [H] sink if sufficient energy and carbon are provided. We examined the effects of inhibiting methanogenesis with 9, 10-anthraquione (AQ) on mixed rumen batch cultures growing on cellulose or starch as sources of energy and carbon contrasting in fermentability, with ammonium (NH4+) or trypticase (Try) as nitrogen (N) sources. Inhibiting methanogenesis with AQ inhibited digestion with cellulose but not with starch, and decreased propionate and increased butyrate molar percentages with both substrates. Inhibiting methanogenesis with 9, 10-anthraquinone increased de novo synthesis of microbial AA with starch but not with cellulose. The decrease in the recovery of [H] caused by the inhibition of methanogenesis was more moderate with starch due to an enhancement of butyrate and AA as [H] sinks. There may be an opportunity to simultaneously decrease the emissions of CH4 and N with some ruminant diets and replace plant protein supplements with less expensive non-protein nitrogen sources such as urea.

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De novo synthesis of trideuteromethyl esters of amino acids for use in GC–MS and GC-tandem MS exemplified for ADMA in human plasma and urine: Standardization, validation, comparison and proof of evidence for their aptitude as internal standards

Journal of Chromatography B ◽

10.1016/j.jchromb.2009.01.005 ◽

2009 ◽

Vol 877 (23) ◽

pp. 2308-2320 ◽

Cited By ~ 21

Author(s):

Dimitrios Tsikas

Keyword(s):

Amino Acids ◽

Human Plasma ◽

De Novo ◽

Tandem Ms ◽

De Novo Synthesis ◽

Internal Standards ◽

Human Plasma And Urine

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Ammonia toxicity under hyponatremic conditions in astrocytes: De novo synthesis of amino acids for the osmoregulatory response

Neurochemistry International ◽

10.1016/j.neuint.2005.04.005 ◽

2005 ◽

Vol 47 (1-2) ◽

pp. 39-50 ◽

Cited By ~ 17

Author(s):

C ZWINGMANN ◽

D LEIBFRITZ

Keyword(s):

Amino Acids ◽

De Novo ◽

Ammonia Toxicity ◽

De Novo Synthesis

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The Uncommon Active Site of D-Amino Acid Transaminase from Haliscomenobacter hydrossis: Biochemical and Structural Insights into the New Enzyme

Molecules ◽

10.3390/molecules26165053 ◽

2021 ◽

Vol 26 (16) ◽

pp. 5053

Author(s):

Alina K. Bakunova ◽

Alena Yu. Nikolaeva ◽

Tatiana V. Rakitina ◽

Tatiana Y. Isaikina ◽

Maria G. Khrenova ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Structural Analysis ◽

Active Site ◽

Active Sites ◽

Structural Basis ◽

Active Site Residues ◽

Type Iv ◽

Fold Type ◽

Arginine Residues

Among industrially important pyridoxal-5’-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes.

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The effects of anticonvulsant compounds on 4-aminopyridine-induced de novo synthesis of neurotransmitter amino acids in rat hippocampus in vitro

Epilepsy Research ◽

10.1016/0920-1211(94)00071-4 ◽

1995 ◽

Vol 20 (2) ◽

pp. 113-120 ◽

Cited By ~ 18

Author(s):

Izet M Kapetanovic ◽

Wayne D Yonekawa ◽

Harvey J Kupferberg

Keyword(s):

Amino Acids ◽

De Novo ◽

Rat Hippocampus ◽

De Novo Synthesis ◽

Neurotransmitter Amino Acids

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Transmembrane transport and intracellular kinetics of amino acids in human skeletal muscle

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1995.268.1.e75 ◽

1995 ◽

Vol 268 (1) ◽

pp. E75-E84 ◽

Cited By ~ 84

Author(s):

G. Biolo ◽

R. Y. Fleming ◽

S. P. Maggi ◽

R. R. Wolfe

Keyword(s):

Skeletal Muscle ◽

Amino Acids ◽

Protein Synthesis ◽

Amino Acid ◽

Human Skeletal Muscle ◽

De Novo ◽

Transmembrane Transport ◽

Acid Transport ◽

De Novo Synthesis ◽

Intracellular Amino Acid

We have used stable isotopic tracers of amino acids to measure in vivo transmembrane transport of phenylalanine, leucine, lysine, alanine, and glutamine as well as the rates of intracellular amino acid appearance from proteolysis, de novo synthesis, and disappearance to protein synthesis in human skeletal muscle. Calculations were based on data obtained by the arteriovenous catheterization of the femoral vessels and muscle biopsy. We found that the fractional contribution of transport from the bloodstream to the total intracellular amino acid appearance depends on the individual amino acid, varying between 0.63 +/- 0.02 for phenylalanine and 0.22 +/- 0.02 for alanine. Rates of alanine and glutamine de novo synthesis were approximately eight and five times their rate of appearance from protein breakdown, respectively. The model-derived rate of protein synthesis was highly correlated with the same value calculated by means of the tracer incorporation technique. Furthermore, amino acid transport rates were in the range expected from literature values. Consequently, we conclude that our new model provides a valid means of quantifying the important aspects of protein synthesis, breakdown, and amino acid transport in human subjects.

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