Two distinct pathways for thymidylate (dTMP) synthesis in (hyper ...

Thermophiles 2003

Two distinct pathways for thymidylate (dTMP) synthesis in (hyper)thermophilic Bacteria and Archaea D. Leduc*, S. Graziani†, L. Meslet-Cladiere*, A. Sodolescu†, U. Liebl† and H. Myllykallio*1 *Institut de Gen ´ etique ´ et Microbiologie, Universite´ de Paris-Sud, 91405 Orsay, France, and †INSERM U451-Laboratory of Optics and Biosciences, Ecole Polytechnique, 91128 Palaiseau, France

Abstract 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.

Introduction Two differences in their chemical groups distinguish the building blocks of RNA from those of DNA. While the sugar of RNA nucleotides corresponds to ribose, the sugar portion of DNA consists of deoxyribose, lacking a hydroxy group in position 2 of ribose. Moreover, DNA contains thymidine, a methylated derivative of the pyrimidine uracil. It is widely believed that these chemical modifications in DNA are necessary for the accurate maintenance of large DNA genomes found in all known free-living organisms. Until recently, thymidylate synthase ThyA was considered the only enzyme capable of catalysing de novo formation of dTMP (thymidylate), an essential DNA precursor. However, strikingly, comparative genomics has revealed a large number of microbial genomes apparently lacking known pathways for formation of dTMP either de novo or by salvaging of thymidine from growth medium. In this contribution, we describe how our combined in silico and experimental approaches led to the unexpected discovery of a new metabolic pathway that operates in DNA precursor synthesis in numerous microbial species.

Discovery of flavin-dependent thymidylate synthase ThyX Despite the fact that Pyrococcus abyssi, a hyperthermophilic anaerobic archeon, is unable to salvage thymidine from Key words: DNA evolution, flavoprotein, hyperthermophile, thymidylate synthase. Abbreviations used: dTMP, thymidylate; H4 MPT, tetrahydromethanopterin. 1

To whom correspondence should be addressed (e-mail hannu.myllykallio@igmors. u-psud.fr).

growth medium [1], we could not detect the thymidylate synthase gene thyA in Pyrococcus species genome sequences. This observation strongly suggested the presence of an uncharacterized enzyme capable of synthesizing dTMP de novo in Pyrococcus sp., as well as in a large number of non-symbiotic microbes. Two independent observations gave possible clues for a functional identification of this putative enzyme. First, in 1989 a gene thy1 of unknown function had been implicated in the thymidine metabolism of Dictyostelium discoideum using genetic complementation tests [2]. Secondly, detailed in silico analyses indicated that the phylogenetic distribution patterns of thyA (classical thymidylate synthase) and thy1 (which we named thyX to avoid confusion with the Thy-1 cell-surface antigen) are almost complementary [1,3], suggesting that ThyA and ThyX proteins could be functionally analogous. We were subsequently able to prove experimentally that ThyX proteins indeed posses a flavindependent thymidylate synthase activity both in vivo and in vitro [1]. Biochemical [1] and structural [4] data have demonstrated that the catalytically relevant forms of Helicobacter pylori and Thermotoga maritima ThyX proteins are formed by four identical subunits (Figure 1A). The observation that the ThyX homotetramer contains four tightly bound FAD molecules supported well the FAD-dependent mechanism for dTMP synthesis we proposed earlier for ThyX proteins. No sequence or structural homology exists between ThyA and ThyX proteins, thus our work firmly established that two distinct classes of thymidylate synthases operate in the microbial world. Moreover, the structure of the T. maritima protein does not show any significant structural similarity to any other known protein, thus indicating that ThyX
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Figure 1 ThyX structure and domain organization (A) Structure of Thermotoga maritima ThyX protein [4]. The structure is formed by four identical subunits that are indicated by different colours. Two different views of the homotetramer are shown. (B) Various domain organizations for ThyX proteins. While the vast majority of thyX genes encode a monomeric ThyX domain, in a few cases this domain is duplicated. The ThyX protein from Trichodesmium erythraeum IMS101 contains HintN and HintC domains that are often found in proteins undergoing protein splicing. The scale represents the number of amino acid residues.

proteins have a novel fold. Although the vast majority of ThyX proteins are composed of approx. 200–250 amino acid residues and presumably form homotetramers in solution, the ThyX homologues for instance from Thermoplasma and Chlamydia species are much longer, as they actually contain two fused ThyX domains in the same polypeptide (Figure 1B). Sequence alignments suggested that these duplication events have taken place several times independently. Whether these duplicated ThyX variants form dimers containing four ThyX modules is unknown. Finally, at least the ThyX protein from Trichodesmium erythraeum IMS101 contains HintN and HintC domains that are often found in proteins undergoing protein splicing (Figure 1B), thus suggesting a plausible mechanism for the observed domain arrangements in ThyX proteins.
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ThyX and ThyA use different reductive mechanisms Although all experimentally characterized ThyA (EC 2.1.1.45) and ThyX (EC 2.1.1.148) proteins use methylene tetrahydrofolate and dUMP as substrates to catalyse the formation of dTMP, the reductive mechanisms of these enzymes are different. In the reductive methylation reaction catalysed by ThyA [5], methylene tetrahydrofolate functions both as a carbon source and source of reducing equivalents, thus leading to the formation of dihydrofolate as a reaction product (see reaction 1 in Scheme 1). Differently from ThyA proteins, bacterial ThyX proteins contain tightly associated FAD as a cofactor [1,4], and their activity is strictly dependent on reduced pyridine nucleotides.

Thermophiles 2003

Scheme 1 Reactions catalysed by ThyA and ThyX proteins CH2 H4 folate, methylene tetrahydrofolate; H2 folate, dihydrofolate; H4 folate, tetrahydrofolate.

The simplest explanation for these observations is that a flavin-mediated hydride transfer from pyridine nucleotides is required for reduction of the methylene group during ThyXcatalysed dTMP formation (see reaction 2 in Scheme 1). It seems likely that in the ThyX-catalysed reaction, methylene tetrahydrofolate functions only as a carbon source, thus resulting in the formation of tetrahydrofolate as a product of the methylation reaction. Note that many Archaea do not contain tetrahydrofolate but rather carry chemically modified variants of H4 MPT (tetrahydromethanopterin), originally discovered in methanogenic Archaea (for a review, see [6]). These chemical modifications differ among various archaeal species. Nevertheless, in the absence of other candidate molecules these ‘chemically modified folates’ are likely to replace methylene tetrahydrofolate as the methylene donor in reactions 1 and 2 in Archaea.

Hyperthermophilic micro-organisms use preferentially ThyX for dTMP synthesis To investigate whether (hyper)thermophilic microbes utilize preferentially either ThyX or ThyA proteins for dTMP synthesis, we have investigated their respective phylogenetic distributions among thermophiles (Table 1). Interestingly, our analyses using 15 completed genome sequences indicate that thyX can be found in approx. 70% of organisms thriving at temperatures
60◦ C (Table 1). In addition, ThyX proteins can be found in thermophilic Bacteria, Euryarchaeota, Crenarchaeota and at least in one double-stranded DNA virus infecting hyperthermophilic Archaea (G. Vestergaard and D. Prangishvili, personal communication), while the distribution of ThyA among thermophiles is much narrower. These observations strongly indicate that thymidylate synthase ThyX is the principal dTMP-producing enzyme in (hyper)thermophilic microbes. The presence of ThyA homologues in some archaeal species is noteworthy (Table 1). Archaeoglobus, Methanocaldococcus, Methanopyrus and Methanothermobacter species all contain a homologue of ThyA [7] and use

Table 1 Distribution of thymidylate synthases ThyX and ThyA in (hyper)thermophilic microbes with a complete genome sequence Growth temperature (◦ C) Euryarchaeota Pyrococcus horikoshii

ThyX

ThyA

96

+

−

Pyrococcus abyssi Pyrococcus furiosus DSM 3638 Thermoplasma acidophilum

96 96 59

+ + +

− − −

Thermoplasma volcanium Archaeoglobus fulgidus DSM Methanocaldococcus jannaschii

60 83 85

+ − −

− + +

110 65

− −

+ +

100 90

+ +

− −

Sulfolobus solfataricus Sulfolobus tokodaii Bacteria

80 80

+ +

− −

Thermotoga maritima Aquifex aeolicus

80 96

+ +

− −

Methanopyrus kandleri AV19 Methanothermobacter thermoautotrophicus Crenarchaeota Pyrobaculum aerophilum Aeropyrum pernix

methylenetetrahydromethanopterin as a carrier of methylene fragments. The methylene/methyl couple is much more electronegative when linked to H4 MPT than in cases where tetrahydrofolate is used as carrier of C1 fragments [6]. Consequently, more reductive power is needed to reduce methylene to methyl when bound to H4 MPT. Moreover, while archaeal ThyX homologues likely use NAD(P)H/FADH2 to reduce methylene, it is unclear whether the ThyA homologues listed in Table 1 use H4 MPT, NAD(P)H/FADH2 or possibly even deazaflavin F420 (a 5 deazaflavin derivative found in methanogenic and sulphatereducing Archaea) as a reductant. The latter ambiguity could explain why, in M. thermoautrophicum, a ThyA protein was found to catalyse only partial reactions for dTMP
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Figure 2 Phylogenetic distributions of thyX and thyA among major prokaryotic groups Dictyostelium discoideum is currently the only known Eukarya with a thyX gene. The universal tree shown is based on the analysis from reference [14]. Distributions were determined using the STRING database and refined using manual Psi-BLAST searches as described previously [1]. The complete analysis used 102 complete genome sequences for cellular organisms. Viral genomes were excluded from the analysis shown here. Note that many Corynebacterinea genomes contain thyX and thyA genes.

synthesis, such as hydrogen exchange and dehalogenation of bromodeoxyuridine [8]. Altogether, these observations suggest that intracellular redox conditions might modulate dTMP synthesis activities of ThyA and/or ThyX proteins.

Phylogenetic distributions of thymidylate synthases in the microbial world To gain further insight into the sporadic phylogenetic distributions of the two thymidylate synthases in cellular organisms, we have now investigated their occurrence in the major bacterial and archaeal groups based upon complete genome information (Figure 2). Recent iterative similarity searches have indicated that ThyX proteins can be found ‘only’ in approx. 35% of completely sequenced microbial genomes (results not shown), suggesting that ThyA proteins play a more widespread role in DNA precursor synthesis. However, this analysis did not account for the considerable bias in completely sequenced genomes. As a matter of fact, almost 60% of the 102 genomes we have analysed correspond
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to different species of α-, β- or γ -proteobacteria and low-GC Gram-positive bacteria (Figure 2 and results not shown). As the vast majority of their genomes contain thyA, the width of ThyX distribution is statistically clearly understated. It is noteworthy that the thyX gene can be found in many photosynthetic, anaerobic or microaerophilic microbes, suggesting that the thyX pathway is preferentially used in species where the NADPH pool is kept relatively reduced. In agreement with this proposal, thyX from Campylobacter jejuni was found to complement an Escherichia coli thyA strain only under microaerophilic conditions [9]. In the light of modern biosynthetic pathways for pyrimidine compounds, uracil-containing DNA (U-DNA) has probably served as an intermediate during the transition from the RNA to the DNA world [10]. Our discovery of a second class of thymidylate synthases has strikingly suggested that the establishment of dTMP-containing DNA (T-DNA) could have taken place twice independently. It is currently unclear whether ThyA or ThyX proteins occurred already in a U-DNA-containing organism. The presence of thyX in (hyper)thermophilic Bacteria, Archaea and Planctomycetes, which have been proposed to correspond to the most deeply

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branching prokaryotes (for Planctomycetes see [11]), may indicate that ThyX could correspond to the ancestral enzyme. It is also of note that a flavin-dependent mechanism for reducing methylene to methyl is used in DNA precursor [1] and protein synthesis [12], as well as for modification of tRNA [13], while the reductive methylation reaction catalysed by ThyA is currently the only known example of its kind.

Conclusions The discovery of a second class of thymidylate synthases using comparative genomics and experimental approaches has provided a striking example of how studies on thermophiles can further our understanding of central metabolic pathways, earlier thought to be universally conserved. In addition, although pathogenic hyperthermophiles or Archaea are yet to be discovered, the presence of thyX in many genomes of pathogenic Bacteria has unexpectedly revealed that studies on thermophiles can help to identify novel targets for antimicrobials.

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