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

Leduc, Damien; Graziani, Sébastien; Meslet‐Cladière, Laurence; Sodolescu, A.; Liebl, Ursula; Myllykallio, Hannu

doi:10.1042/bst0320231

Cited by 36 publications

(31 citation statements)

References 11 publications

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“…ThyX proteins represent a recently discovered family of flavoproteins, essential for DNA replication in at least 30% of the microbial genomes sequenced to date [the considerable biases in available genome sequences underestimate this number (14)]. During this work we have undertaken structure-function studies on the functional roles of conserved ThyX amino acid residues.…”

Section: Discussionmentioning

confidence: 99%

Functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers

Leduc

Graziani

Lipowski

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

103

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Little is known about the catalytic mechanism of the recently discovered ThyX family of flavin-dependent thymidylate synthases that are required for thymidylate (deoxythymidine 5 -monophosphate) synthesis in a large number of microbial species. Using a combination of site-directed mutagenesis and biochemical measurements, we have identified several residues of the Helicobacter pylori ThyX protein with crucial roles in ThyX catalysis. By providing functional evidence that the active site(s) of homotetrameric ThyX proteins is formed by three different subunits, our findings suggest that ThyX proteins have evolved through multimerization of inactive monomers. Moreover, because the active-site configurations of ThyX proteins, present in many human pathogenic bacteria, and of human thymidylate synthase ThyA are different, our results will aid in the identification of compounds specifically inhibiting microbial growth.

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Section: Discussionmentioning

confidence: 99%

Functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers

Leduc

Graziani

Lipowski

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

103

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“…All the Borrelia sp. proteins have the ThyX motif of RHRX 7 S (positions 90 to 100 of B. hermsii ThyX) that is common to these proteins at their conserved amino-terminal domain (30,35). The gene products of B. hermsii and B. burgdorferi share the following residues in common with other ThyX orthologues (B. hermsii ThyX numbering): H65, E70, F76, R90, H91, R92, S100, Y103, E168, R171, L174, P175, L197, R198, E206, R208, A211, and P223 (29).…”

Section: Vol 188 2006mentioning

confidence: 99%

“…In other species of organisms, the thyX product, ThyX, is thymidylate synthase X (EC 2.1.1.148), which catalyzes the methylation of dUMP to yield the DNA precursor dTMP. Sequences homologous to thyX have been found in several groups of bacteria, including cyanobacteria, actinobacteria, and epsilonproteobacteria such as Helicobacter pylori, but they are also present in some double-stranded DNA viruses of bacteria and at least one eukaryote, the slime mold Dictyostelium discoides (30,35). Other groups of bacteria, including gamma-proteobacteria such as Escherichia coli and firmicutes such as Bacillus subtilis, usually have instead a thyA gene, which encodes thymidylate synthase A (ThyA; EC 2.1.1.45) and is not homologous to thyX (11,30).…”

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confidence: 99%

“…Sequences homologous to thyX have been found in several groups of bacteria, including cyanobacteria, actinobacteria, and epsilonproteobacteria such as Helicobacter pylori, but they are also present in some double-stranded DNA viruses of bacteria and at least one eukaryote, the slime mold Dictyostelium discoides (30,35). Other groups of bacteria, including gamma-proteobacteria such as Escherichia coli and firmicutes such as Bacillus subtilis, usually have instead a thyA gene, which encodes thymidylate synthase A (ThyA; EC 2.1.1.45) and is not homologous to thyX (11,30). ThyX proteins are homotetrameric and flavin adenine dinucleotide (FAD) dependent, while ThyA proteins are homodimeric and FAD independent in their catalysis (33,35).…”

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confidence: 99%

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Function and Evolution of Plasmid-Borne Genes for Pyrimidine Biosynthesis inBorreliaspp

et al. 2006

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The thyX gene for thymidylate synthase of the Lyme borreliosis (LB) agent Borrelia burgdorferi is located in a 54-kb linear plasmid. In the present study, we identified an orthologous thymidylate synthase gene in the relapsing fever (RF) agent Borrelia hermsii, located it in a 180-kb linear plasmid, and demonstrated its expression. The functions of the B. hermsii and B. burgdorferi thyX gene products were evaluated both in vivo, by complementation of a thymidylate synthase-deficient Escherichia coli mutant, and in vitro, by testing their activities after purification. The B. hermsii thyX gene complemented the thyA mutation in E. coli, and purified B. hermsii ThyX protein catalyzed the conversion of dTMP from dUMP. In contrast, the B. burgdorferi ThyX protein had only weakly detectable activity in vitro, and the B. burgdorferi thyX gene did not provide complementation in vivo. The lack of activity of B. burgdorferi's ThyX protein was associated with the substitution of a cysteine for a highly conserved arginine at position 91. The B. hermsii thyX locus was further distinguished by the downstream presence in the plasmid of orthologues of nrdI, nrdE, and nrdF, which encode the subunits of ribonucleoside diphosphate reductase and which are not present in the LB agents B. burgdorferi and Borrelia garinii. Phylogenetic analysis suggested that the nrdIEF cluster of B. hermsii was acquired by horizontal gene transfer. These findings indicate that Borrelia spp. causing RF have a greater capability for de novo pyrimidine synthesis than those causing LB, thus providing a basis for some of the biological differences between the two groups of pathogens.Spirochetes are a coherent group of bacteria with a unique morphology and cell structure (38). The point of divergence of spirochetes from other bacterial groups was deep in time, and the topology of spirochetes' branching from other phyla, such as proteobacteria and cyanobacteria, remains unresolved. Phylogenetic inference has been advanced with the availability of genome sequences of pathogenic spirochete species from the following three genera: Borrelia, including Borrelia burgdorferi (13,19) and B. garinii (23), two agents of Lyme borreliosis (LB); Treponema, including Treponema pallidum (20), the agent of syphilis, and Treponema denticola (51), a mouth organism associated with periodontal disease; and two serovars of Leptospira interrogans (36,44). Leptospires have free-living capacity and, not surprisingly, larger chromosomes, at ϳ4.5 Mb, than the ϳ1-Mb chromosomes of the obligately parasitic organisms T. pallidum (20) and B. burgdorferi (18). But the leptospires' branching from the common ancestor of Treponema and Borrelia appears to be nearly as ancient as the spirochetes' origin itself, and for the most part, leptospire sequences have been of limited use for further defining evolution within the spirochete clade.The several Borrelia species can be divided into two major groups on the basis of DNA sequence and biological differences (2). One group includes all the agen...

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“…This observation led to the identification of alternative flavin-dependent thymidylate synthases (FDTSs), which are encoded by the thyX gene and have no sequence or structure homology to classic TSase enzymes (2,6,7). Furthermore, multiple studies have identified key differences in the molecular mechanism of catalysis between FDTSs and classic TSases (2,4,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). These differences, along with the fact that the thyX gene is present in many human pathogens (e.g., bacteria causing Anthrax, Tuberculosis, Typhus, and other diseases), renders these flavo-enzymes as potential targets for rational inhibitor design, possibly affording compounds that might be effective antimicrobial drugs (11,(22)(23)(24).…”

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confidence: 99%

Folate binding site of flavin-dependent thymidylate synthase

Koehn

Perissinotti

Moghram

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The DNA nucleotide thymidylate is synthesized by the enzyme thymidylate synthase, which catalyzes the reductive methylation of deoxyuridylate using the cofactor methylene-tetrahydrofolate (CH 2 H 4 folate). Most organisms, including humans, rely on the thyA-or TYMS-encoded classic thymidylate synthase, whereas, certain microorganisms, including all Rickettsia and other pathogens, use an alternative thyX-encoded flavin-dependent thymidylate synthase (FDTS). Although several crystal structures of FDTSs have been reported, the absence of a structure with folates limits understanding of the molecular mechanism and the scope of drug design for these enzymes. Here we present X-ray crystal structures of FDTS with several folate derivatives, which together with mutagenesis, kinetic analysis, and computer modeling shed light on the cofactor binding and function. The unique structural data will likely facilitate further elucidation of FDTSs' mechanism and the design of structure-based inhibitors as potential leads to new antimicrobial drugs.

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

Cited by 36 publications

References 11 publications

Functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers

Functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers

Function and Evolution of Plasmid-Borne Genes for Pyrimidine Biosynthesis inBorreliaspp

Folate binding site of flavin-dependent thymidylate synthase

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