An Alternative Flavin-Dependent Mechanism for Thymidylate Synthesis Hannu Myllykallio, Gerard Lipowski, Damien Leduc, Jonathan Filee, Patrick Forterre, and Ursula Liebl

Supplementary Material

Materials and Methods
Open reading frames for hypothetical proteins corresponding to P. abyssi (PAB0861) and H. pylori (HP1533) thyX genes, were obtained by PCR, using specific primers and P. abyssi chromosomal DNA and clone GHPEH26 (American Type Culture Collection no. 628507), respectively, as templates. PCR products were cloned into the topoisomerase I-activated pBAD TOPO® TA vector (Invitrogen), permitting tightly regulated gene expression in E. coli. All plasmid clones were confirmed by DNA sequencing. Expected molecular masses of PAB0861 and HP1533, including an amino-terminal translation enhancer and carboxy-terminal V5 and hexahistidine epitopes, are 33.7 and 31.5 kDa, respectively. Protein expression was induced with 0.2% L-arabinose. Expressed proteins were detected using monoclonal V5-specific antibodies (Invitrogen) according to manufacture's recommendations.

Site-directed mutagenesis reactions, confirmed by DNA sequencing, were performed using a QuickChange kit (Stratagene). Two complementary primers (the sequence of only one of these is indicated) were used to change codon Ser107 (indicated in fig. S1 by an asterisk) of H. pylori thyX into alanine and stop codons. The sequences of the mutagenic primers were 5'-GCG-AGC-TTG-AGC-GTG-AAA-GCT-AGC-CGT-TAC-ACT-TTG-AAG-3' (alanine mutation) and 5'-GCG-AGC-TTG-AGC-GTG-AAA-TAA-AGC-CGT-TAC-ACT-TTG-AAG-3' (stop codon mutation).

Biologically active epitope-tagged H. pylori ThyX protein was purified from 200 ml cultures of pGL2/E. coli 2913 (table S2) after 3 hour induction by 0.2% L-arabinose. A QIAexpression kit (Qiagen) under standard native conditions was used for purification as described by the manufacturer, including 10% (volume/volume) glycerol in all buffers. Obtained protein samples were dialyzed against 50 mM phosphate buffer, pH 7.4, 10% (volume/volume) glycerol after elution to remove imidazol. Protein concentration in pure samples was determined by a dye reagent protein assay (Biorad) using bovine serum albumin as standard. Using high-pressure liquid chromatography as earlier described (1), we showed that the co-factor released from H. pylori ThyX eluted with a retention time 3.3 min. Retention times of 3.2 min and 3.8 min were observed for FAD and FMN standards, respectively, suggesting that H. pylori ThyX protein associates with FAD in vivo. Different enzyme preparations of H. pylori ThyX contained 0.4 - 0.5 molecules of flavin per monomer.

Tritium release assays with purified enzyme were performed as described in Table 2. Reactions were stopped after 60 minutes at 37°C by two extractions with 250 l of activated charcoal [10% (weight/volume) Norit A] in 2% trichloroacetic acid to remove radioactive nucleotides from reaction mixtures. Radioactivity remaining in the supernatant was determined as described (2). The dTMP forming activity of H. pylori ThyX was measured under similar conditions, except 1 M [6-³H]dUMP (specific activity 21.2 Ci/mmol, Moravek Biochemicals) together with 200 M non-radioactive dUMP was used, including 10% glycerol in the reaction. Neutralized perchloric acid extracts of reaction mixtures were analyzed by high pressure liquid chromatography. A Beckman Ultrasphere ODS (4.6 mm x 25 cm) reverse phase C18 column was used to separate the reaction product (dTMP) from an excess of the substrate (dUMP). Isocratic elution with 10 mM phosphate buffer, pH 4.0 (1 ml/min) was used (dUMP, retention time 5.6 min; dTMP, 10.2 min).

References and Notes
1. C. D. Capo-Chichi et al., Am. J. Clin. Nutr. 71, 978 (2000).
2. H. Myllykallio et al., Science 288, 2212 (2000).

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Supplemental Figure 1. Sequence alignment of a diverse set of ThyX homologs was obtained manually. The conserved <<ThyX motif>> and the Ser residue (asterisk) altered by site-directed mutagenesis (table S1) are indicated. Poorly aligned amino- and carboxy-terminal regions were removed from the alignment.

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Supplemental Table 1. H. pylori thyX complements E. coli 2913 into thymidine prototrophy.
Plasmid	Relevant characteristics	Phenotype1
		M9 + Amp + arabinose	M9 + Amp	M9 + Amp + thymidine
2913 (thyA)
pGL1	P. abyssi thyX on pBAD-Topo®, AmpR	-	-	++++
pGL2	H. pylori thyX on pBAD-Topo®, AmpR	+++2	-	++++
pGL3	As pGL2, Ser107Ala	-	-	++++
pGL4	As pGL2, Ser107Stop	-	-	++++

¹Thymidine independent growth of a recombination deficient E. coli 2913 (complete genotype "thyA752 recA56" referred to "thyA" in this work), carrying various plasmids, was scored either in the presence of 0.2% L-arabinose or 50 g/ml thymidine using M9 minimal medium supplemented with biotin, leucine, proline, thiamine and glucose. Ampicillin (Amp) was used at 100 g/ml. -, No growth visible after 5 days; +++, colonies (1 mm in diameter) appeared after 4 days; ++++, colonies (1 mm in diameter) appeared within 3 days.
²Growth was inhibited only by high concentration of trimethoprim (100 g/ml) in the absence of thymidine. Under similar conditions, 10 g/ml of trimethoprim was sufficient to inhibit the growth of wild-type E. coli strains.