Identification of a novel gene encoding a flavin-dependent tRNA:m(5)U methyltransferase in bacteria—evolutionary implications

Formation of 5-methyluridine (ribothymidine) at position 54 of the T-psi loop of tRNA is catalyzed by site-specific tRNA methyltransferases (tRNA:m(5)U-54 MTase). In all Eukarya and many Gram-negative Bacteria, the methyl donor for this reaction is S-adenosyl-l-methionine (S-AdoMet), while in several Gram-positive Bacteria, the source of carbon is N(5), N(10)-methylenetetrahydrofolate (CH(2)H(4)folate). We have identified the gene for Bacillus subtilis tRNA:m(5)U-54 MTase. The encoded recombinant protein contains tightly bound flavin and is active in Escherichia coli mutant lacking m(5)U-54 in tRNAs and in vitro using T7 tRNA transcript as substrate. This gene is currently annotated gid in Genome Data Banks and it is here renamed trmFO. TrmFO (Gid) orthologs have also been identified in many other bacterial genomes and comparison of their amino acid sequences reveals that they are phylogenetically distinct from either ThyA or ThyX class of thymidylate synthases, which catalyze folate-dependent formation of deoxyribothymine monophosphate, the universal DNA precursor

Flavin-dependent thymidylate synthase X limits chromosomal DNA replication

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Expression and purification of untagged and histidine-tagged folate-dependent tRNA:m5U54 methyltransferase from Bacillus subtilis

International audienceFolate-dependent tRNA m5U methyltransferase TrmFO is a flavoprotein that catalyzes the C5-methylation of uridine at position 54 in the T-Psi-C loop of tRNA in several bacteria. Here we report the cloning and optimization of expression in Escherichia coli BL21 (DE3) of untagged, N-terminus, C-terminus (His)6-tagged TrmFO from Bacillus subtilis. Tagged and untagged TrmFO were purified to homogeneity by metal affinity or ion exchange and heparin affinity, respectively, followed by size-exclusion chromatography. The tag did not significantly alter the expression level, flavin content, activity and secondary structure of the protein. Cop. 2010 Elsevier Inc. All rights reserved

In vitro detection of the enzymatic activity of folate-dependent tRNA (Uracil-54,-C5)-methyltransferase: evolutionary implications.

Formation of 5-methyluridine (ribothymidine) at position 54 of the T-psi loop of tRNA is catalyzed by site-specific tRNA methyltransferases (tRNA[uracil-54,C5]-MTases). In eukaryotes and many bacteria, the methyl donor for this reaction is generally S-adenosyl-L-methionine (S-AdoMet). However, in other bacteria, like Enterococcus faecalis and Bacillus subtilis, it was shown that the source of carbon is N(5),N(10)-methylenetetrahydrofolate (CH(2)=THF). Recently we have determined that the Bacillus subtilis gid gene (later renamed to trmFO) encodes the folate-dependent tRNA(uracil-54,C5)-MTase. Here, we describe a procedure for overexpression and purification of this recombinant enzyme, as well as detection of its activity in vitro. Inspection of presently available sequenced genomes reveals that trmFO gene is present in most Firmicutes, in all alpha- and delta-Proteobacteria (except Rickettsiales in which the trmFO gene is missing), Deinococci, Cyanobacteria, Fusobacteria, Thermotogales, Acidobacteria, and in one Actinobacterium. Interestingly, trmFO is never found in genomes containing the gene trmA coding for S-adenosyl-L-methionine-dependent tRNA (uracil-54,C5)-MTase. The phylogenetic analysis of TrmFO sequences suggests an ancient origin of this enzyme in bacteria

Flavin-Dependent Thymidylate Synthase ThyX Activity: Implications for the Folate Cycle in Bacteria▿ †

Although flavin-dependent ThyX proteins show thymidylate synthase activity in vitro and functionally complement thyA defects in heterologous systems, direct proof of their cellular functions is missing. Using insertional mutagenesis of Rhodobacter capsulatus thyX, we constructed the first defined thyX inactivation mutant. Phenotypic analyses of the obtained mutant strain confirmed that R. capsulatus ThyX is required for de novo thymidylate synthesis. Full complementation of the R. capsulatus thyX::spec strain to thymidine prototrophy required not only the canonical thymidylate synthase ThyA but also the dihydrofolate reductase FolA. Strikingly, we also found that addition of exogenous methylenetetrahydrofolate transiently inhibited the growth of the different Rhodobacter strains used in this work. To rationalize these experimental results, we used a mathematical model of bacterial folate metabolism. This model suggests that a very low dihydrofolate reductase activity is enough to rescue significant thymidylate synthesis in the presence of ThyX proteins and is in agreement with the notion that intracellular accumulation of folates results in growth inhibition. In addition, our observations suggest that the presence of flavin-dependent thymidylate synthase X provides growth benefits under conditions in which the level of reduced folate derivatives is compromised

Insights into folate/FAD-dependent tRNA methyltransferase mechanism: role of two highly conserved cysteines in catalysis.

International audienceThe flavoprotein TrmFO methylates specifically the C5 carbon of the highly conserved uridine 54 in tRNAs. Contrary to most methyltransferases, the 1-carbon unit transferred by TrmFO derives from 5,10-methylenetetrahydrofolate and not from S-adenosyl-L-methionine. The enzyme also employs the FAD hydroquinone as a reducing agent of the C5 methylene U54-tRNA intermediate in vitro. By analogy with the catalytic mechanism of thymidylate synthase ThyA, a conserved cysteine located near the FAD isoalloxazine ring was proposed to act as a nucleophile during catalysis. Here, we mutated this residue (Cys-53 in Bacillus subtilis TrmFO) to alanine and investigated its functional role. Biophysical characterization of this variant demonstrated the major structural role of Cys-53 in maintaining both the integrity and plasticity of the flavin binding site. Unexpectedly, gel mobility shift assays showed that, like the wild-type enzyme, the inactive C53A variant was capable of forming a covalent complex with a 5-fluorouridine-containing mini-RNA. This result confirms the existence of a covalent intermediate during catalysis but rules out a nucleophilic role for Cys-53. To identify the actual nucleophile, two other strictly conserved cysteines (Cys-192 and Cys-226) that are relatively far from the active site were replaced with alanine, and a double mutant C53A/C226A was generated. Interestingly, only mutations that target Cys-226 impeded TrmFO from forming a covalent complex and methylating tRNA. Altogether, we propose a revised mechanism for the m(5)U54 modification catalyzed by TrmFO, where Cys-226 attacks the C6 atom of the uridine, and Cys-53 plays the role of the general base abstracting the C5 proton

Functional analysis of FAD-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1

International audienceSequence analysis of the 330-kb double-stranded DNA genome of Paramecium bursaria chlorella virus-1 revealed an open reading frame A674R that encodes a protein with up to 53% amino acid identity to a recently discovered new class of thymidylate synthases, called ThyX. Unlike the traditional thymidylate synthase, ThyA, that uses methylenetetrahydrofolate (CH(2)H(4)folate) as both a source of the methylene group and the reductant, CH(2)H(4)folate only supplies the methylene group in ThyX-catalyzed reactions. Furthermore, ThyX only catalyzes thymidylate (dTMP) formation in the presence of reduced pyridine nucleotides and oxidized FAD. The distribution and transcription patterns of the a674r gene in Chlorella viruses were examined. The a674r gene was cloned, and the protein was expressed in Escherichia coli. Biochemical characterization of the P. bursaria chlorella virus-1 recombinant ThyX protein indicates that it is more efficient at converting dUMP to dTMP than previously studied ThyX enzymes, thus allowing more detailed mechanistic studies of the enzyme. The ThyX-dUMP complexes with bound FAD function as efficient NAD(P) H oxidases, indicating that dUMP binds to the enzyme prior to NAD(P) H. This oxidation activity is directly linked to FAD reduction. Our results indicate that ThyX-specific inhibitors can be designed that do not affect ThyA enzymes. Finally, a model is proposed for the early stages of ThyX catalysis

Structural and functional insight into serine hydroxymethyltransferase from Helicobacter pylori.

Serine hydroxymethyltransferase (SHMT), encoded by the glyA gene, is a ubiquitous pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the formation of glycine from serine. The thereby generated 5,10-methylene tetrahydrofolate (MTHF) is a major source of cellular one-carbon units and a key intermediate in thymidylate biosynthesis. While in virtually all eukaryotic and many bacterial systems thymidylate synthase ThyA, SHMT and dihydrofolate reductase (DHFR) are part of the thymidylate/folate cycle, the situation is different in organisms using flavin-dependent thymidylate synthase ThyX. Here the distinct catalytic reaction directly produces tetrahydrofolate (THF) and consequently in most ThyX-containing organisms, DHFR is absent. While the resulting influence on the folate metabolism of ThyX-containing bacteria is not fully understood, the presence of ThyX may provide growth benefits under conditions where the level of reduced folate derivatives is compromised. Interestingly, the third key enzyme implicated in generation of MTHF, serine hydroxymethyltransferase (SHMT), has a universal phylogenetic distribution, but remains understudied in ThyX-containg bacteria. To obtain functional insight into these ThyX-dependent thymidylate/folate cycles, we characterized the predicted SHMT from the ThyX-containing bacterium Helicobacter pylori. Serine hydroxymethyltransferase activity was confirmed by functional genetic complementation of a glyA-inactivated E. coli strain. A H. pylori ΔglyA strain was obtained, but exhibited markedly slowed growth and had lost the virulence factor CagA. Biochemical and spectroscopic evidence indicated formation of a characteristic enzyme-PLP-glycine-folate complex and revealed unexpectedly weak binding affinity of PLP. The three-dimensional structure of the H. pylori SHMT apoprotein was determined at 2.8Ǻ resolution, suggesting a structural basis for the low affinity of the enzyme for its cofactor. Stabilization of the proposed inactive configuration using small molecules has potential to provide a specific way for inhibiting HpSHMT