RESUMO
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.
Assuntos
Aldeído Liases/metabolismo , Di-Hidropteroato Sintase/metabolismo , Difosfotransferases/metabolismo , Complexos Multienzimáticos/metabolismo , Saccharomyces cerevisiae/enzimologia , Tetra-Hidrofolatos/metabolismo , Aldeído Liases/análise , Aldeído Liases/genética , Proteínas de Ligação a DNA/genética , Di-Hidropteroato Sintase/análise , Di-Hidropteroato Sintase/genética , Difosfotransferases/análise , Difosfotransferases/genética , Escherichia coli/genética , Deleção de Genes , Teste de Complementação Genética , Hifas/genética , Hifas/crescimento & desenvolvimento , Glicoproteínas de Membrana , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Complexos Multienzimáticos/análise , Complexos Multienzimáticos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Arabidopsis/química , Sítios de Ligação , Calnexina/química , Proteínas de Transporte/química , Clonagem Molecular , Cristalografia por Raios X , DNA/química , Proteínas de Drosophila/química , Drosophila melanogaster , Eletroforese em Gel de Poliacrilamida , Proteínas de Membrana/química , Metaloproteínas/química , Metionina/química , Modelos Moleculares , Dados de Sequência Molecular , Oligonucleotídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Selenometionina/química , Homologia de Sequência de AminoácidosRESUMO
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',6,7-13C5]dihydroneopterin was treated with aldolase in deuterated buffer. The resulting, partially deuterated [6alpha,6,7-13C3]6-hydroxymethyl-7,8-dihydropterin was converted to partially deuterated 6-(R)-[6,7,9,11-13C4]5,10-methylenetetrahydropteroate by a sequence of three enzyme-catalyzed reactions followed by treatment with [13C]formaldehyde. The product was analyzed by multinuclear NMR spectroscopy. The data show that the carbinol group of enzymatically formed 6-hydroxymethyl-dihydropterin contained 2H predominantly in the pro-S position.