Biosynthesis of Tetrahydrofolate

Abstract

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...

A Disconnect between High-Affinity Binding and Efficient Regulation by Antifolates and Purines in the Tetrahydrofolate Riboswitch

“…In our original study, we obtained THF as a mixture of the 6 S and 6 R epimers, whereas in biology THF exists exclusively in the 6 S conformation (Illarionova et al, 2002). This mixture yielded a 2:1 THF:RNA binding stoichiometry with no indications that the RNA was discriminating between the two epimers.…”

Identification of Growth Inhibitors of the Fish Pathogen Saprolegnia parasitica Using in silico Subtractive Proteomics, Computational Modeling, and Biochemical Validation

“…Sulfonamides are predicted to interact with the protein SPRG_19504 in S. parasitica , which belongs to the dihydropteroate synthase (DHPS) family and contains a 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (HPPK) domain and a pterin-binding domain. In several bacteria and lower eukaryotes, HPPK (EC 2.7.6.3) and DHPS (EC 2.5.1.15) enzymes catalyze sequential reactions in the folic acid biosynthetic pathway ( Illarionova et al, 2002 ). Higher eukaryotes obtain folate from dietary sources and lack necessary enzymes for folate biosynthesis ( Matherly, 2001 ), whereas eubacteria and lower eukaryotes are able to synthesize tetrahydrofolate.…”

Genome-scale metabolic network analysis and drug targeting of multi-drug resistant pathogen Acinetobacter baumannii AYE

“…4F); hence, essential metabolites consumed by any such homologous enzymes were further removed. As a result of this step in EMFilter, a total of 9 final essential metabolites were selected; they are 2-amino-4hydroxy-6-hydroxymethyl-7,8-dihydropteridine (AHHMP), 39,40 D-glutamate (DGLU), 41,42 2,3-dihydrodipicolinate (DHDP), 43,44 2-amino-4-hydroxy-6-(D-erythro-1,2,3-trihydroxypropyl)-7,8-dihydropteridine (DHP), 45 3-dehydroshikimate (DHSK), [46][47][48] 1-deoxy-D-xylulose 5-phosphate (DX5P), 49,50 3-dehydroquinate (DQT), 51,52 2-dehydro-3-deoxy-D-octonate (KDO) 53,54 and 4-aminobenzoate (PABA) 55,56 (Table 3). The enzymes involved in the outgoing reactions around these essential metabolites were predicted as final drug target candidates.…”