Hannu Myllykallio1, Damien Leduc1, Jonathan Filee1 and Ursula Liebl2 1Institut de Ge´ne´tique et Microbiologie CNRS UMR8621; Universite´ de Paris-Sud, Orsay, France2Institut National de la Sante´ et de la Recherche Me´dicale U451; Laboratory of Optics and Biosciences, Ecole Polytechnique-ENSTA,Palaiseau, France Reduced folate derivatives participate in numerous comparative genomics has revealed a large number of reactions of bacterial intermediary metabolism. Conse- microbial species apparently lacking genes encoding quently, the well-characterized enzyme implicated in either one of these enzymes (see ). The absence of thyA in these genomes was explained by our recent finding reductase FolA – was considered to be essential for that a large family of previously uncharacterized ThyX bacterial growth. However, comparative genomics has (also known as Thy1) proteins corresponds to a novel class revealed several bacterial genome sequences that of flavin-dependent thymidylate synthases . As thyA appear to lack the folA gene. Here, we provide in silico and thyX genes have, with few exceptions, mutually evidence indicating that folA-lacking bacteria use a exclusive phylogenetic distributions , and the novel recently discovered class of flavin-dependent thymidy- class of thymidylate synthases is present in up to 30% of late synthases for deoxythymidine-50-monophosphate completed microbial genome sequences these data synthesis, and propose that many bacteria must con- unequivocally demonstrate that two major pathways for tain uncharacterized sources for reduced folate mol- dTMP formation operate in the microbial world.
ecules that are still waiting to be discovered.
Although both ThyA and ThyX are CH2H4folate- dependent enzymes, the two distinct classes of thymidy- One-carbon units linked to tetrahydrofolate (H4folate) are late synthases appear to differ markedly regarding their required for RNA-, DNA- and protein-synthesis (In reductive mechanisms Our data indicate that, actively dividing cells, a large quantity of reduced folates is unlike ThyA proteins, Helicobacter pylori ThyX uses required for synthesis of deoxythymidine-50-monophosphate CH2H4folate as only a one-carbon donor, whereas the (dTMP or thymidylate), a unique nucleotide component ofDNA. Consequently, dTMP synthesis has the potential to diminish the pool-size of reduced folates, thus havingindirect consequences for other branches of the intermediarymetabolism. Until recently, the only known pathway for de novo synthesis of thymidylate was by thymidylate synthaseThyA, an enzyme methylating deoxyuridine-50-monophos- phate (dUMP). Uniquely for a biological reaction, the reductive methylation of dUMP by ThyA is intrinsically ThyA uses methylenetetrahydrofolate (CH2H4folate) as both carbon source and reductant H2folate formedthrough oxidation of tetrahydrofolate (H4folate) by ThyA israpidly reduced by FolA, as only reduced folate derivatives are functional in intermediary metabolism Takinginto account the pivotal functional importance of ThyA andFolA, both of these enzymes have been used widely as targets for compounds that inhibit cellular proliferation.
For instance, bacterial FolA is specifically inhibited bytrimethoprim, a clinically relevant antibacterial agent .
Fig. 1. All cells need reduced folate cofactors for the biosyntheses of many com-pounds. The de novo pathway for folate compounds directly synthesizes dihydro- Folate metabolism in bacteria carrying a novel folate, although only tetrahydrofolate can serve as a donor of one-carbon units forDNA-, RNA-, co-factor- and protein-syntheses Trimethoprim acts as a specific inhibitor of bacterial dihydrofolate reductase (FolA). The red arrow indicates that, In contrast to the earlier prediction that functional in actively dividing cells, the majority of tetrahydrofolate derivatives are used for coupling of ThyA and FolA should be vital for all bacteria, dTMP synthesis. Abbreviations: dTMP, deoxythymidine monophosphate; GTP,guanosine triphosphate; H2folate, dihydrofolate; H4folate, tetrahydrofolate; Met,methionine; Met-tRNAfMet, a tRNA bearing N-formyl methionine that is required Corresponding author: Hannu Myllykallio (
for initiation of protein synthesis in bacteria.
0966-842X/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0966-842X(03)00101-X Table 1. Bacterial species using thymidylate synthase ThyX for thymidylate synthesisaa aNote that the majority of species included in this non-comprehensive list seemingly lack FolA. Bacterial species relevant for human health are indicated in bold.
Abbreviations: FolA, bacterial dihydrofolate reductase; Tdk, thymidine kinase required for a salvage of extracellular thymidine; ThyA and ThyX, flavin-dependent and‘canonical’ thymidylate synthase, respectively.
bArchaea containing chemically modified folates were excluded from the list.
cPhylogenetic distributions were determined using COG and STRING databases. The symbols þ and 2 refer to the presence and absence of a given gene in a genome,respectively.
dIf not otherwise indicated, data were collected using the publicly available sequence data accessible at and .
eSee for phylogenetic analysis of these FolA sequences.
electrons required for formation of the methyl moiety are not have an absolute requirement for FolA in their folate transferred from reduced pyridine nucleotides via an metabolism. However, bacteria lacking folA must still enzyme-bound flavin cofactor to form dTMP This contain reduced folates for RNA and protein syntheses to reaction mechanism for dTMP formation maintains the take place, although their source for reduced folates folate in its reduced form (as H4folate) at the end of the catalytic cycle. Therefore, the proposed difference inthe reductive mechanisms of ThyA and ThyX offers a Alternative pathway(s) for H4folate formation? plausible explanation as to why all thyA-containing One explanation for the absence of folA in a wide bacteria contain folA, despite that this gene is often absent phylogenetic range of bacteria would be the presence of from thyX-containing organisms. Surprisingly, this obser- alternative bacterial pathways and/or enzymes forming vation also indicates that thyX-containing organisms do H4folate. The existence of such alternative pathways was Fig. 2. The different reductive mechanisms of thymidylate synthases (a) ThyA and (b) ThyX. ThyA proteins use methylenetetrahydrofolate (CH2H4folate) both as carbon andelectron source, thus resulting in the formation of H2folate. Reduced flavin nucleotides (FADH2) have an obligatory role in ThyX catalysis , thus strongly indicating thatdTMP catalysis is linked – differently from ThyA proteins – to formation of H4folate. Strikingly, although all thyA-carrying bacteria also contain dihydrofolate reductasefolA, the known pathways for formation of H4folate are absent in several thyX-containing bacteria (), suggesting the presence of an alternative dihydrofolatereductase in many bacterial species. Note also that an enzyme implicated in generation of CH2H4folate – serine transhydroxymethylase (SHT) – has a universal phyloge-netic distribution. Abbreviations: CH2H4folate, methylenetetrahydrofolate; DHFR?, a postulated alternative dihydrofolate reductase; dTMP, deoxythymidine monophos-phate; dUMP, deoxyuridine monophosphate; FADþ, flavin adenine dinucleotide (oxidized form); FADH2, flavin adenine dinucleotide (reduced form); Gly, glycine; H2folate,dihydrofolate; H4folate, tetrahydrofolate; Ser, serine; ThyA and ThyX, thymidylate synthase A and X, respectively.
Clostridium acetobutylicum (Firmicutes)
Clostridium perfringens (Firmicutes)
Ralstonia solanacearum Thermotoga maritima
Rickettsia conorii (α-proteobacteria)
Chlamydophila pneumoniae
Chlamydia muridarum
Chlamydia trachomatis
Fig. 3. Phylogenetic analysis of various FolA sequences. The maximum likelihood analysis was performed as described in the text Bacterial species indicated in red cor-respond to species containing FolA and ThyX proteins. The presence of FolA in ThyX-containing Rickettsia conorii and Clostridium spp. could result from a lateral genetransfer event. The tree was rooted using eukaryotic sequences. Abbreviation: Tn, transposon.
suspected earlier, but before the discovery of ThyX, their reductase (accession number gi:78392) identifies this physiological relevance was largely ignored. For instance, protein as oxygen-insensitive NAD(P)H nitroreductase E. coli strains carrying inactive folA are still viable and (NfsB). As genetic data have revealed a role for NfsB in contain reduced folates . Direct evidence for this mediating microbial resistance to several nitro-substi- unexpected source of H4folate in E. coli DfolA strains is tuted compounds it is possible that this enzyme also lacking, although it has been proposed to result from the plays a role in folate metabolism, although this is presence of an alternative dihydrofolate reductase in this species. The identity of this putative enzyme is unclear, Helicobacter spp. and Campylobacter spp. that lack folA although it could correspond to an E. coli enzyme that was provide particularly interesting cases in the experimental originally described as ‘dihydropteridine reductase’, which identification of new, physiologically relevant pathways is able to reduce in vitro H2folate to H4folate, albeit very leading to the formation of H4folate. Not only do H. pylori inefficiently . In our opinion, this low level of dihydro- and C. jejuni use thymidylate synthase ThyX for folate reductase activity is not sufficient to explain the dTMP synthesis, but they are also considered endogen- surprisingly high amount of reduced folates in E. coli DfolA ously resistant to low levels of trimethoprim, a classical strains (60 – 80% compared with that found for wild-type inhibitor of bacterial FolA The molecular basis for strains ). In addition, we have now noticed that the this chromosomally encoded trimethoprim resistance of N-terminal protein sequence of E. coli dihydropteridine 1-proteobacteria is poorly understood. However, salvage of thymidine compounds from the growth medium is unlikely explanation for the unexpected observation that life to contribute to trimethoprim resistance, and genomic without two ‘essential’ enzymes – ThyA and FolA – is data (not shown) indicate the absence of the trimetho- still possible. Further understanding of the hitherto poorly prim-insensitive plasmid-encoded family 2 dihydrofolate characterized folate metabolism in bacteria using ThyX for reductase in these species. Taken together, these thymidylate synthesis will undoubtedly aid in under- observations suggest that the uncharacterized enzyme, standing the evolution of intermediary metabolism, as well as in designing new compounds for inhibiting synthesizes H4folate in 1-proteobacteria and possibly in Interestingly, a subgroup of thyX-containing organisms also contains the folA gene (), revealing that We would like to thank Patrick Forterre for helpful discussions, and Julian Eaton-Rye and Stephane Skouloubris for critical comments on the arily mutually exclusive, assuming that FolA has not functionally replaced the predicted alternative dihydrofo-late reductase. The simultaneous presence of thyX and 1 Carreras, C.W. and Santi, D.V. (1995) The catalytic mechanism folA in a given genome could result either from the non- and structure of thymidylate synthase. Annu. Rev. Biochem. 64, orthologous replacement of thyA by thyX, or, alternatively, from transfer of folA into a thyX-containing organism.
2 Huovinen, P. et al. (1995) Trimethoprim and sulfonamide resistance.
Phylogenetic analysis of FolA () supports this latter Antimicrob. Agents Chemother. 39, 279 – 289 possibility. In particular, our data indicate that the 3 Myllykallio, H. et al. (2002) An alternative flavin-dependent mechan- position of Rickettsia conorii FolA is the result of lateral ism for thymidylate synthesis. Science 297, 105 – 107 4 Galperin, M.Y. and Koonin, E.V. (2000) Who’s your neighbor? New gene transfer, because its position is far away from all the computational approaches for functional genomics. Nat. Biotechnol.
other a-proteobacteria. They further indicate that FolA in Clostridium spp. is closely related to a FolA variant 5 Herrington, M.B. and Chirwa, N.T. (1999) Growth properties of a encoded by an E. coli plasmid (Unfortunately, the folA null mutant of Escherichia coli K12. Can. J. Microbiol. 45, phylogenetic positions of Chlamydia spp. and Thermotoga 6 Hamm-Alvarez, S.F. et al. (1990) The presence and distribution of maritima have not yet been firmly established, thus reduced folates in Escherichia coli dihydrofolate reductase mutants.
preventing a firm conclusion regarding the origin of their folA genes. It is also worth noting that the transfer 7 Vasudevan, S.G. et al. (1992) Dihydropteridine reductase from of a transposon-coded folA into clinical isolates of C. jejuni Escherichia coli exhibits dihydrofolate reductase activity. Biol.
8 Koziarz, J.W. et al. (1998) Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nsfB in development of resistance to ThyX and alternative dihydrofolate reductase as ideal 5-nitrofuran derivatives in Escherichia coli. J. Bacteriol. 180, For several reasons, ThyX proteins and the postulated 9 Giladi, M. et al. (2002) Genetic evidence for a novel thymidylate alternative dihydrofolate reductase are ideal targets for synthase in the halophilic archaeon Halobacterium salinarum and in compounds specifically inhibiting microbial growth. Not Campylobacter jejuni. FEMS Microbiol. Lett. 216, 105 – 109 10 Gibreel, A. and Sko¨ld, O. (1998) High-level resistance to trimethoprim only is thyX present in several pathogenic bacteria lacking in clinical isolates of Campylobacter jejuni by acquisition of foreign folA genes and absent in humans, but also the de novo genes (dfr1 and dfr9) expressing drug-insensitive dihydrofolate synthesis of pyrimidine compounds is required for the reductases. Antimicrob. Agents Chemother. 42, 3059 – 3064 growth and/or virulence of pathogenic microorganisms 11 Fields, P.I. et al. (1986) Mutants of Salmonella typhirium that cannot that often reside in pyrimidine-limited environments survive within the macrophage are avirulent. Proc. Natl. Acad. Sci.
The development of ThyX inhibitors will be 12 Ko¨hler, S. et al. (2002) The analysis of the intramacrophagic virulome facilitated by the recently solved structure of the Thermo- of Brucella suis deciphers the environment encountered by the toga maritima ThyX protein which has already pathogen inside the macrophage host cell. Proc. Natl. Acad. Sci.
revealed that the two classes of thymidylate synthases are completely unrelated structurally. Therefore, the design of 13 Kuhn, P. et al. (2002) Crystal structure of Thy1, a thymidylate ThyX inhibitors does not have to rely on small structural synthase complementing protein from Thermotoga maritima at 2.25 A differences between ThyX and human ThyA proteins.
14 Matthews, R.G. (1996) One carbon metabolism. In Escherichia Coli Similarly, it is tempting to speculate that yet-to-be- and Salmonella – Cellular and Molecular Biology (Neidhardt, F.C., identified alternative pathways and/or enzymes partici- 15 Van de Casteele, M. et al. (1995) The dihydrofolate reductase-encoding be unrelated to their functional counterparts in Eukarya.
gene dyrA of the hyperthermophilic bacterium Thermotoga maritima.
Gene 158, 101 – 105 16 Tatusov, R.L. et al. (1997) A genomic perspective on protein families.
The discovery of an alternative flavin-dependent mechan- 17 von Mering, C. et al. (2003) STRING: a database of predicted functional ism for thymidylate synthesis has revealed a plausible associations between proteins. Nucleic Acids Res. 31, 258 – 261



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