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Tetrahydromonapteriu is a major pterin in Escherichia coli and is hypothesized to be the cofactor for phenylalanine hydroxylase (PhhA) in Pseudomonas aeruginosa, but neither its biosynthetic origin nor its cofactor role has been clearly demonstrated. A comparative genomics analysis implicated the enigmaticfo/X and folM genes in tetrahydromonapterin synthesis via their phyletic distribution and chromosomal clustering patterns, folX encodes dihydroneopterin triphosphate epimerase, which interconverts dihydroneopterin triphosphate and dihydromonapterin tripbosphate, folM encodes an unusual short-chain dehydrogenase/ reductase known to have dihydrofolate and dihydrobiopterin reductase activity. The roles of FolX and FolM were tested experimentally first in E. coli, which lacks PhhA and in which the expression of P. aeruginosa PhhA plus the recycling enzyme pterin 4a-carbinolamine dehydratase, PhhB, rescues tyrosine auxotrophy. This rescue was abrogated by deletingfo/X orfoiM and restored by expressing the deleted gene from a plasmid. The folX deletion selectively eliminated tetrahydromonapterin production, which far exceeded folate production. Purified FolM showed high, NADPH-dependent dihydromonapterin reductase activity. These results were substantiated in P. aeruginosa by deleting tyrA (making PhhA the sole source of tyrosine) and folX. The [DELTA]tyrA strain was, as expected, prototrophic for tyrosine, whereas the [DELTA]tyrA [DELTA]folX strain was auxotrophic. As in E. coli, the foiX deletant lacked tetrahydromonapterin. Collectively, these data establish that tetrahydromonapterin formation requires both FolX and FolM, that tetrahydromonapterin is the physiological cofactor for PhhA, and that tetrahydromonapterin can outrank folate as an end product of pterin biosynthesis. doi: 10.1128/JB.01198-09

Dihydroneopterin aldolase (FolB) catalyzes conversion of dihydroneopterin to 6-hydroxymethyldihydropterin (HMDHP) in the classical folate biosynthesis pathway. However, folB genes are missing from the genomes of certain bacteria from the phyla Chloroflexi, Acidobacteria, Firmicutes, Planctomycetes, and Spirochaetes. Almost all of these folB-deficient genomes contain an unusual paralog of the tetrahydrohiopterin synthesis enzyme 6-pyruvoyltetrahy. dropterin synthase (PTPS) in which a glutamate residue replaces or accompanies the catalytic cysteine. A similar PTPS paralog from the malaria parasite Plasmodium falciparum is known to form HMDHP from dihydroneopterin triphosphate in vitro and has been proposed to provide a bypass to the FolB step in vivo. Bacterial genes encoding PTPS-like proteins with active-site glutamate, cysteine, or both residues were accordingly tested together with the P. falciparum gene for complementation of the Escherichia coli folB mutation. The P. falciparum sequence and bacterial sequences with glutamate or glutamate plus cysteine were active; those with cysteine alone were not. These results demonstrate that PTPS paralogs with an active-site glutamate (designated PTPS-III proteins) can functionally replace FolB in vivo. Recombinant bacterial PTPS-III proteins, like the P. falciparum enzyme, mediated conversion of dihydroneopterin triphosphate to HMDHP, but other PTPS proteins did not. Neither PTPS-III nor other PTPS proteins exhibited significant dihydroneopterin aldolase activity. Phylogenetic analysis indicated that PTPS-III proteins may have arisen independently in various PTPS lineages. Consistent with this possibility, merely introducing a glutamate residue into the active site of a PTPS protein conferred incipient activity in the growth complementation assay, and replacing glutamate with alanine in a PTPS-III protein abolished complementation.