Comparative genomics guided discovery of two missing archaeal enzyme families involved in the biosynthesis of the pterin moiety of tetrahydromethanopterin and tetrahydrofolate

Abstract

C-1 carriers are essential cofactors in all domains of life, and in Archaea, these can be derivatives of tetrahydromethanopterin (H(4)-MPT) or tetrahydrofolate (H(4)-folate). Their synthesis requires 6-hydroxymethyl-7,8-dihydropterin diphosphate (6-HMDP) as the precursor, but the nature of pathways that lead to its formation were unknown until the recent discovery of the GTP cyclohydrolase IB/MptA family that catalyzes the first step, the conversion of GTP to dihydroneopterin 2',3'-cyclic phosphate or 7,8-dihydroneopterin triphosphate [El Yacoubi, B.; et al. (2006) J. Biol. Chem., 281, 37586-37593 and Grochowski, L. L.; et al. (2007) Biochemistry46, 6658-6667]. Using a combination of comparative genomics analyses, heterologous complementation tests, and in vitro assays, we show that the archaeal protein families COG2098 and COG1634 specify two of the missing 6-HMDP synthesis enzymes. Members of the COG2098 family catalyze the formation of 6-hydroxymethyl-7,8-dihydropterin from 7,8-dihydroneopterin, while members of the COG1634 family catalyze the formation of 6-HMDP from 6-hydroxymethyl-7,8-dihydropterin. The discovery of these missing genes solves a long-standing mystery and provides novel examples of convergent evolutions where proteins of dissimilar architectures perform the same biochemical function.

Figure 1

Early steps of tetrahydrofolate and tetrahydromethanopterin pathways in Bacteria and Archaea. Most bacteria use the FolE (or FolE2)/FolB/FolK route (in blue) to 6-HMDP even if some use the bacterial PTPS-III shunt (in green). Several routes to the common 6-HMDP intermediate in tetrahydrofolate and tetrahydromethanopterin are found in Archaea. A common pathway is the FolE2/MptD/MptE route (in red) such as in H. volcanii paralleling the bacterial pathway. However, some methanogens such as M. jannaschii use the MptA/MptB/MptD/MptE route, whereas P. furiosus uses the archaeal PTPS-III shunt. Phosphatases still to be identified are noted by a question mark (?). FolE/FolE2, GTP cyclohydrolase IA/IB (GCYH-IA/B); FolB, 7,8-dihydroneopterin aldolase (DHNA); FolK, 7,8-dihydro-6-hydroxymethylpterin diphosphokinase (6-HMDPK); MptA, archaeal GTP cyclohydrolase I (Fe(II)-dependent enzyme); MptB, Fe(II) dependent-cyclic phosphodiesterase; MptD, archaeal specific DHNA; MptE, archaeal specific 6-HMDPK; PTPS-III/PTPS-V/PTPS-VI, pyruvoyltetrahydropterin synthase paralogs involved in 6-HMDP synthesis.

Figure 2

Phylogenetic distribution of predicted 6-HMDP synthesis genes in a subset of archaeal genomes. The presence of a symbol denotes the presence of a member of the protein family represented in that specific column in the genome covered in the corresponding line. Symbols and corresponding protein family are in the same color. Abbreviations have been defined in the Figure 1 legend. 6-HMDP synthesis genes might still be unidentified in organisms highlighted in yellow. Organisms highlighted in beige are most certainly pterin auxotrophs. Symbols linked by a line represent fused proteins. The full analysis is available in the Public SEED database in the Subsystem: “Pterin Biosynthesis Archaea”.

Figure 3

Comparative genomic evidence. (A) Clustering of COG1634 and COG2098 genes with pterin and cofactor biosynthetic related genes. Abbreviation not found in the text: FolP-like, dihydropteroate synthase-like enzyme homologous to the bacterial folate enzyme FolP but of unknown function; MptG, β-ribofuranosylaminobenzene 5′-phosphate synthase; FolM, alternative dihydrofolate reductase; F420-lig, coenzyme F420-0: l-glutamate ligase. (B) The archaeal DHNA (MptD) tetramer with bound pterin ring mimic (PDB 2OGF). The individual subunits of MJ0408 are shown with differently colored cartoons, the bound ligand 8-oxoguanine with orange carbons. (C) Putative active site of the archaeal DHNA with manually docked neopterin. The MptD structure is from PDB 2IEC, and the neopterin ligand (orange carbons) is from PDB 2O90 (in alternative conformation B). The active site residues contributed by three different subunits are shown with green, cyan, and magenta carbons, respectively (as in panel B and Supplemental Figure 3).

Figure 4

Experimental validations. (A) The MG1655 ΔfolB::Kan^R strain (VDC3276) was transformed with the empty vector pBAD24 (top), pfolB_Ec (middle), or pMJ0408 (bottom). The resulting strains were plated on LB (with appropriate antibiotics), LB supplemented with arabinose (0.2%), or LB supplemented with dT and grown for 48 h. (B) The C600 ΔfolK::tetB strain was transformed with empty plasmid pBAD24 (top), pfolK_Ec (middle), or pMJ1634 (bottom). The resulting strains were plated on LB (with appropriate antibiotics) with or without dT supplementation and grown for 48 h. In both cases, complementation of the dT auxotrophy phenotypes by the archaeal clones were observed even in the absence of the inducer, arabinose. (C) Purified MJ0408 derived protein was incubated with H₂Neo as described in the methods section. After incubation, the sample was oxidized with iodine to convert the dihydropterins to the fluorescent pterins and assayed by HPLC with fluorescence detection. The first peak to elute at ∼10 min was neopterin, and the second at ∼16.5 min was 6-hydroxymethylpterin. The figure shows an assay where about half of the substrate was converted into product. No product was observed at zero time or in an assay run without added enzyme. The MonoQ fraction of the purified enzyme produced from an E. coli extract not expressing the MJ0408 derived protein likewise did not show any activity. (D) Purified PF0930 derived protein was incubated with 6-HMD and ATP as described in the methods section. After incubation, the sample was oxidized with iodine to convert the dihydropterins to the fluorescent pterins and assayed by HPLC with fluorescence detection. The first peak to elute at ∼5.2 min was 6-hydroxymethylpterin-PP, the second at ∼6.6 min was 6-hydroxymethylpterin-P, and the third at 16.5 min was 6-hydroxymethylpterin. The figure shows an assay where about 90% of the substrate was converted into product. The origin of the 6-hydroxymethylpterin-P is not clear but could arise from the hydrolysis of the 6-hydroxymethylpterin-PP during sample preparation. No product was observed at zero time or in an assay run without added enzyme. The MonoQ fraction of the purified enzyme produced from an E. coli extract not expressing the PF0930 derived protein likewise did not show any activity. Similar results were obtained with the MJ1634 protein.