Tetrahydrofolate (vitamin B9) and its folate derivatives are essential cofactors in one-carbon (C1) transfer reactions and absolutely required for the synthesis of a variety of different compounds including methionine and purines. Most plants, microbial eukaryotes, and prokaryotes synthesize folate de novo. We have characterized an important enzyme in this pathway, the Saccharomyces cerevisiae FOL1 gene. Expression of the budding yeast gene FOL1 in Escherichia coli identified the folate biosynthetic enzyme activities dihydroneopterin aldolase (DHNA), 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (HPPK), and dihydropteroate synthase (DHPS). All three enzyme activities were also detected in wild-type yeast strains, whereas fol1Delta deletion strains only showed background activities, thus demonstrating that Fol1p catalyzes three sequential steps of the tetrahydrofolate biosynthetic pathway and thus is the central enzyme of this pathway, which starting from GTP consists of seven enzymatic reactions in total. Fol1p is exclusively localized to mitochondria as shown by fluorescence microscopy and immune electronmicroscopy. FOL1 is an essential gene and the nongrowth phenotype of the fol1 deletion leads to a recessive auxotrophy for folinic acid (5'-formyltetrahydrofolate). Growth of the fol1Delta deletion strain on folinic acid-supplemented rich media induced a dimorphic switch with haploid invasive and filamentous pseudohyphal growth in the presence of glucose and ammonium, which are known suppressors of filamentous and invasive growth. The invasive growth phenotype induced by the depletion of C1 carrier is dependent on the transcription factor Ste12p and the flocullin/adhesin Flo11p, whereas the filamentation phenotype is independent of Ste12p, Tec1p, Phd1p, and Flo11p, suggesting other signaling pathways as well as other adhesion proteins.

The folding topology, quaternary structure and amino acid sequence of epimerase is similar to that of the dihydroneopterin aldolase involved in the biosynthesis of the vitamin folic acid. The monomer fold of epimerase is also topologically similar to that of GTP cyclohydrolase I (GTP CH-1), 6-pyrovoyl tetrahydropterin synthase (PTPS) and uroate oxidase (UO). Despite a lack of significant sequence homology these proteins share a common subunit fold and oligomerize to form central beta barrel structures employing different cyclic symmetry elements, D4, D5, D3 and D2, respectively. Moreover, these enzymes have a topologically equivalent acceptor site for the 2-amino-4-oxo pyrimidine (2-oxo-4-oxo pyrimidine in uroate oxidase) moiety of their respective substrates.

An open reading frame located at 69.0 kilobases on the Escherichia coli chromosome was shown to code for dihydroneopterin aldolase, catalyzing the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin in the biosynthetic pathway of tetrahydrofolate. The gene was subsequently designated folB. The FolB protein shows 30% identity to the paralogous dihydroneopterin-triphosphate epimerase, which is specified by the folX gene located at 2427 kilobases on the E. coli chromosome. The folX and folB gene products were both expressed to high yield in recombinant E. coli strains, and the recombinant proteins were purified to homogeneity. Both enzymes form homo-octamers. Aldolase can use L-threo-dihydroneopterin and D-erythro-dihydroneopterin as substrates for the formation of 6-hydroxymethyldihydropterin, but it can also catalyze the epimerization of carbon 2 of dihydroneopterin and dihydromonapterin at appreciable velocity. Epimerase catalyzes the epimerization of carbon 2 in the triphosphates of dihydroneopterin and dihydromonapterin. However, the enzyme can also catalyze the cleavage of the position 6 side chain of several pteridine derivatives at a slow rate. Steady-state kinetic parameters are reported for the various enzyme-catalyzed reactions. We propose that the polarization of the 2-hydroxy group of the substrate could serve as the initial reaction step for the aldolase as well as for the epimerase activity. A deletion mutant obtained by targeting the folX gene of E. coli has normal growth properties on complete medium as well as on minimal medium. Thus, the physiological role of the E. coli epimerase remains unknown. The open reading frame ygiG of Hemophilus influenzae specifies a protein with the catalytic properties of an aldolase. However, the genome of H. influenzae does not specify a dihydroneopterin-triphosphate epimerase.

The moaABC operon of Escherichia coli is involved in early steps of the biosynthesis of the molybdenum-binding cofactor molybdopterin, but the precise functions of the cognate proteins are not known. The crystal structure of the MoaB protein from E. coli was determined by multiple anomalous dispersion at 2.1 angstroms A resolution and refined to an R factor of 20.4% (Rfree = 25.0%). The protein is a 32-symmetric hexamer, with the monomers consisting of a central beta-sheet flanked by helices on both sides. The overall fold of the monomer is similar to those of the MogA protein of E. coli, the G-domains of rat and human gephyrin and the G-domains of Cnx1 protein from A. thaliana, all of which are involved in the insertion of an unknown molybdenum species into molybdopterin to form the molybdenum cofactor. Furthermore, the MoaB protein shows significant sequence similarity to the cinnamon protein from Drosophila melanogaster. In addition to other functions, all these proteins are involved in the biosynthesis of the molybdenum cofactor and have been shown to bind molybdopterin. The close structural homology to MogA and the gephyrin and Cnx1 domains suggests that MoaB may bind a hitherto unidentified pterin compound, possibly an intermediate in molybdopterin biosynthesis.

7,8-Dihydroneopterin aldolase catalyzes the formation of the tetrahydrofolate precursor, 6-hydroxymethyl-7,8-dihydropterin, and is a potential target for antimicrobial and anti-parasite chemotherapy. The last step of the enzyme-catalyzed reaction is believed to involve the protonation of an enol type intermediate. In order to study the stereochemical course of that reaction step, [1,2,3,6,7-13 C 5 ]dihydroneopterin was treated with aldolase in deuterated buffer. The resulting, partially deuterated [6␣,6,7-13 C 3 ]6-hydroxymethyl-7,8-dihydropterin was converted to partially deuterated 6-(R)-[6,7,9,11-13 C 4 ]5,10-methylenetetrahydropteroate by a sequence of three enzyme-catalyzed reactions followed by treatment with [13 C]formaldehyde. The product was analyzed by multinuclear NMR spectroscopy. The data show that the carbinol group of enzymatically formed 6-hydroxymethyl-dihydropterin contained 2 H predominantly in the pro-S position.Tetrahydrofolate and its derivatives are essential cofactors of one-carbon metabolism. Although plants and many microorganisms obtain folate coenzymes by de novo synthesis, vertebrates are absolutely dependent on nutritional sources (1). Insufficient supply of the vitamin is conducive to anemia in adults and to neural tube malformation in human embryos (2).The biosynthesis of tetrahydrofolate has been studied in some detail (for review see Ref.3). The first committed step catalyzed by GTP cyclohydrolase I converts GTP into dihydroneopterin triphosphate (1, Fig. 1). The triphosphate motif is removed by a previously unknown process, and the resulting 7,8-dihydro-D-neopterin (2) is converted to 6-hydroxymethyldihydropterin (3) by dihydroneopterin aldolase (FolB) (4). The consecutive action of FolK, FolP, FolC, and FolA enzymes finally affords 6-(S)-tetrahydrofolate via the intermediates 4 -6.The folate biosynthetic pathway is a well established drug target for antimicrobial as well as antiparasite therapy. Sulfonamides, the first synthetic antimicrobial and antiparasitic drugs with a broad action spectrum, act via inhibition of dihydropteroate synthase, the penultimate enzyme of the dihydrofolate biosynthetic pathway (Fig. 1, step E) (5-7), and trimethoprim acts against a variety of bacterial pathogens via inhibition of dihydrofolate reductase (Fig. 1, step G) (8 -10).The rapid development of microbial resistance against all antibiotics in current use has generated an urgent requirement for novel anti-infective agents. Because the folate pathway is already a well established drug target, it appears worthwhile to explore other folate biosynthetic enzymes besides dihydropteroate synthase and dihydrofolate reductase. This paper describes studies on the mechanism of dihydroneopterin aldolase. C 5 ]GTP was prepared as described (11). All other reagents used were of the highest purity available. A Nucleosil C18 HPLC 1 column (4 ϫ 250 mm) was from Schambeck, Bad Honnef, Germany. Superdex 75, Superdex 200, Q-Sepharose Fast Flow, and DEAE-Sepharose Fast Flow were purchased from Amersham Bioscienc...