Two distinct pathways for thymidylate (dTMP) synthesis in (hyper)thermophilic Bacteria and Archaea

D. Leduc; S. Graziani; L. Meslet-Cladiere; A. Sodolescu; U. Liebl; H. Myllykallio

doi:10.1042/bst0320231

Two distinct pathways for thymidylate (dTMP) synthesis in (hyper)thermophilic Bacteria and Archaea

Biochemical Society Transactions ◽

10.1042/bst0320231 ◽

2004 ◽

Vol 32 (2) ◽

pp. 231-235 ◽

Cited By ~ 25

Author(s):

D. Leduc ◽

S. Graziani ◽

L. Meslet-Cladiere ◽

A. Sodolescu ◽

U. Liebl ◽

...

Keyword(s):

Thymidylate Synthase ◽

De Novo ◽

Thermophilic Bacteria ◽

De Novo Synthesis ◽

Microbial World ◽

Alternative Pathways ◽

Thymidylate Synthases ◽

Micro Organisms ◽

Similarity Searches ◽

Pyrococcus Abyssi

The hyperthermophilic anaerobic archaeon Pyrococcus abyssi, which lacks thymidine kinase, incorporates label from extracellular uracil, but not from thymidine, into its DNA. This implies that P. abyssi must synthesize dTMP (thymidylate), an essential precursor for DNA synthesis, de novo. However, iterative similarity searches of the three completed Pyrococcus genomes fail to detect candidate genes for canonical thymidylate synthase ThyA, suggesting the presence of alternative pathways for dTMP synthesis. Indeed, by identifying a novel class of flavin-dependent thymidylate synthases, ThyX, we have recently proven that two distinct pathways for de novo synthesis of dTMP are operational in the microbial world. While both thyX and thyA can be found in hyperthermophilic micro-organisms, the phylogenetic distribution of thyX among hyperthermophiles is wider than that of thyA. In this contribution, we discuss the differences in the distinct mechanisms of dTMP synthesis, with a special emphasis on hyperthermophilic micro-organisms.

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An enzymatic activation of formaldehyde for nucleotide methylation

Nature Communications ◽

10.1038/s41467-021-24756-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Charles Bou-Nader ◽

Frederick W. Stull ◽

Ludovic Pecqueur ◽

Philippe Simon ◽

Vincent Guérineau ◽

...

Keyword(s):

Amino Acids ◽

Thymidylate Synthase ◽

Active Site ◽

De Novo ◽

Crystallographic Structure ◽

De Novo Synthesis ◽

Active Site Residues ◽

Enzymatic Activation ◽

Enzyme Cofactors ◽

Unique Ability

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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Alternative Pathways for Siroheme Synthesis inKlebsiella aerogenes

Journal of Bacteriology ◽

10.1128/jb.183.1.328-335.2001 ◽

2001 ◽

Vol 183 (1) ◽

pp. 328-335 ◽

Cited By ~ 9

Author(s):

M. Miriam Kolko ◽

Lori A. Kapetanovich ◽

Jeffrey G. Lawrence

Keyword(s):

Biosynthetic Pathway ◽

De Novo ◽

Enteric Bacteria ◽

Enteric Bacterium ◽

De Novo Synthesis ◽

Alternative Pathways ◽

E Coli ◽

Sulfur Starvation ◽

Nitrite Reductases ◽

Coenzyme B

ABSTRACT Siroheme, the cofactor for sulfite and nitrite reductases, is formed by methylation, oxidation, and iron insertion into the tetrapyrrole uroporphyrinogen III (Uro-III). The CysG protein performs all three steps of siroheme biosynthesis in the enteric bacteriaEscherichia coli and Salmonella enterica. In either taxon, cysG mutants cannot reduce sulfite to sulfide and require a source of sulfide or cysteine for growth. In addition, CysG-mediated methylation of Uro-III is required for de novo synthesis of cobalamin (coenzyme B12) in S. enterica. We have determined that cysG mutants of the related enteric bacterium Klebsiella aerogenes have no defect in the reduction of sulfite to sulfide. These data suggest that an alternative enzyme allows for siroheme biosynthesis in CysG-deficient strains ofKlebsiella. However, Klebsiella cysG mutants fail to synthesize coenzyme B12, suggesting that the alternative siroheme biosynthetic pathway proceeds by a different route. Gene cysF, encoding an alternative siroheme synthase homologous to CysG, has been identified by genetic analysis and lies within the cysFDNC operon; the cysF gene is absent from the E. coli and S. entericagenomes. While CysG is coregulated with the siroheme-dependent nitrite reductase, the cysF gene is regulated by sulfur starvation. Models for alternative regulation of the CysF and CysG siroheme synthases in Klebsiella and for the loss of thecysF gene from the ancestor of E. coli andS. enterica are presented.

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Targeting Methyltransferases in Human Pathogenic Bacteria: Insights into Thymidylate Synthase (TS) and Flavin-Dependent TS (FDTS)

Molecules ◽

10.3390/molecules24081638 ◽

2019 ◽

Vol 24 (8) ◽

pp. 1638 ◽

Cited By ~ 1

Author(s):

Cecilia Pozzi ◽

Ludovica Lopresti ◽

Giusy Tassone ◽

Stefano Mangani

Keyword(s):

Thymidylate Synthase ◽

Pathogenic Bacteria ◽

De Novo ◽

Functional Characterization ◽

Mechanisms Of Action ◽

Human Pathogenic Bacteria ◽

Thymidylate Synthases ◽

Open Debate ◽

Definition Of

In cells, thymidylate synthases provide the only de novo source of 2′-deoxythymidine-5′-monophosphate (dTMP), required for DNA synthesis. The activity of these enzymes is pivotal for cell survival and proliferation. Two main families of thymidylate synthases have been identified in bacteria, folate-dependent thymidylate synthase (TS) and flavin-dependent TS (FDTS). TS and FDTS are highly divergent enzymes, characterized by exclusive catalytic mechanisms, involving different sets of cofactors. TS and FDTS mechanisms of action have been recently revised, providing new perspectives for the development of antibacterial drugs targeting these enzymes. Nonetheless, some catalytic details still remain elusive. For bacterial TSs, half-site reactivity is still an open debate and the recent evidences are somehow controversial. Furthermore, different behaviors have been identified among bacterial TSs, compromising the definition of common mechanisms. Moreover, the redox reaction responsible for the regeneration of reduced flavin in FDTSs is not completely clarified. This review describes the recent advances in the structural and functional characterization of bacterial TSs and FDTSs and the current understanding of their mechanisms of action. Furthermore, the recent progresses in the development of inhibitors targeting TS and FDTS in human pathogenic bacteria are summarized.

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Mechanistic and structural basis for inhibition of thymidylate synthase ThyX

Open Biology ◽

10.1098/rsob.120120 ◽

2012 ◽

Vol 2 (10) ◽

pp. 120120 ◽

Cited By ~ 22

Author(s):

Tamara Basta ◽

Yap Boum ◽

Julien Briffotaux ◽

Hubert F. Becker ◽

Isabelle Lamarre-Jouenne ◽

...

Keyword(s):

Crystal Structure ◽

Thymidylate Synthase ◽

Active Site ◽

De Novo ◽

Tight Binding ◽

Binding Pocket ◽

Structural Basis ◽

Microbial Genomes ◽

Thymidylate Synthases ◽

Thymidylate Synthesis

Nature has established two mechanistically and structurally unrelated families of thymidylate synthases that produce de novo thymidylate or dTMP, an essential DNA precursor. Representatives of the alternative flavin-dependent thymidylate synthase family, ThyX, are found in a large number of microbial genomes, but are absent in humans. We have exploited the nucleotide binding pocket of ThyX proteins to identify non-substrate-based tight-binding ThyX inhibitors that inhibited growth of genetically modified Escherichia coli cells dependent on thyX in a manner mimicking a genetic knockout of thymidylate synthase. We also solved the crystal structure of a viral ThyX bound to 2-hydroxy-3-(4-methoxybenzyl)-1,4-naphthoquinone at a resolution of 2.6 Å. This inhibitor was found to bind within the conserved active site of the tetrameric ThyX enzyme, at the interface of two monomers, partially overlapping with the dUMP binding pocket. Our studies provide new chemical tools for investigating the ThyX reaction mechanism and establish a novel mechanistic and structural basis for inhibition of thymidylate synthesis. As essential ThyX proteins are found e.g. in Mycobacterium tuberculosis and Helicobacter pylori , our studies have also potential to pave the way towards the development of new anti-microbial compounds.

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Characterization of Monocyte-Associated Factor V

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649565 ◽

1993 ◽

Vol 70 (02) ◽

pp. 273-280 ◽

Cited By ~ 5

Author(s):

Janos Kappelmayer ◽

Satya P Kunapuli ◽

Edward G Wyshock ◽

Robert W Colman

Keyword(s):

Monoclonal Antibody ◽

Light Chain ◽

Peripheral Blood ◽

De Novo ◽

Factor V ◽

Blood Monocytes ◽

De Novo Synthesis ◽

Hep G2 ◽

Hep G2 Cells ◽

Peripheral Blood Monocytes

SummaryWe demonstrate that in addition to possessing binding sites for intact factor V (FV), unstimulated peripheral blood monocytes also express activated factor V (FVa) on their surfaces. FVa was identified on the monocyte surface by monoclonal antibody B38 recognizing FVa light chain and by human oligoclonal antibodies H1 (to FVa light chain) and H2 (to FVa heavy chain) using immunofluorescence microscopy and flow cytometry. On Western blots, partially cleaved FV could be identified as a 220 kDa band in lysates of monocytes. In addition to surface expression of FVa, monocytes also contain intracellular FV as detected only after permeabilization by Triton X-100 by monoclonal antibody B10 directed specifically to the Cl domain not present in FVa. We sought to determine whether the presence of FV in peripheral blood monocytes is a result of de novo synthesis.Using in situ hybridization, no FV mRNA could be detected in monocytes, while in parallel control studies, factor V mRNA was detectable in Hep G2 cells and CD18 mRNA in monocytes. In addition, using reverse transcriptase and the polymerase chain reaction, no FV mRNA was detected in mononuclear cells or in U937 cells, but mRNA for factor V was present in Hep G2 cells using the same techniques. These data suggest that FV is present in human monocytes, presumably acquired by binding of plasma FV, and that the presence of this critical coagulation factor is not due to de novo synthesis.

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Platelets Stimulate Thromboplastin Synthesis in Human Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657323 ◽

1983 ◽

Vol 49 (02) ◽

pp. 069-072 ◽

Cited By ~ 20

Author(s):

U L H Johnsen ◽

T Lyberg ◽

K S Galdal ◽

H Prydz

Keyword(s):

Endothelial Cells ◽

De Novo ◽

Umbilical Vein ◽

Time Dependent ◽

De Novo Synthesis ◽

Human Endothelial Cells ◽

Tumor Promotor ◽

Coagulation Assay ◽

Platelet Aggregates ◽

Umbilical Vein Endothelial Cells

SummaryHuman umbilical vein endothelial cells in culture synthesize thromboplastin upon stimulation with phytohaemagglutinin (PHA) or the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The thromboplastin activity is further strongly enhanced in a time dependent reaction by the presence of gel-filtered platelets or platelet aggregates. This effect was demonstrable at platelet concentrations lower than those normally found in plasma, it may thus be of pathophysiological relevance. The thromboplastin activity increased with increasing number of platelets added. Cycloheximide inhibited the increase, suggesting that de novo synthesis of the protein component of thromboplastin, apoprotein III, is necessary.When care was taken to remove monocytes no thromboplastin activity and no apoprotein HI antigen could be demonstrated in suspensions of gel-filtered platelets, platelets aggregated with thrombin or homogenized platelets when studied with a coagulation assay and an antibody neutralization technique.

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