RESUMEN
The structure of a putative protease from Bacteroides thetaiotaomicron features an unprecedented binding site for flavin mononucleotide. The flavin isoalloxazine ring is sandwiched between two tryptophan residues in the interface of the dimeric protein. We characterized the recombinant protein with regard to its affinity for naturally occurring flavin derivatives and several chemically modified flavin analogs. Dissociation constants were determined by isothermal titration calorimetry. The protein has high affinity to naturally occurring flavin derivatives, such as riboflavin, FMN, and FAD, as well as lumichrome, a photodegradation product of flavins. Similarly, chemically modified flavin analogs showed high affinity to the protein in the nanomolar range. Replacement of the tryptophan by phenylalanine gave rise to much weaker binding, whereas in the tryptophan to alanine variant, flavin binding was abolished. We propose that the protein is an unspecific scavenger of flavin compounds and may serve as a storage protein in vivo.
Asunto(s)
Proteínas Bacterianas/química , Bacteroides/enzimología , Mononucleótido de Flavina/química , Flavina-Adenina Dinucleótido/química , Péptido Hidrolasas/química , Riboflavina/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteroides/genética , Mononucleótido de Flavina/genética , Mononucleótido de Flavina/metabolismo , Flavina-Adenina Dinucleótido/genética , Flavina-Adenina Dinucleótido/metabolismo , Genómica , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Riboflavina/genética , Riboflavina/metabolismoRESUMEN
In Saccharomyces cerevisiae , thiamin pyrimidine is formed from histidine and pyridoxal phosphate (PLP). The origin of all of the pyrimidine atoms has been previously determined using labeling studies and suggests that the pyrimidine is formed using remarkable chemistry that is without chemical or biochemical precedent. Here we report the overexpression of the closely related Candida albicans pyrimidine synthase (THI5p) and the reconstitution and preliminary characterization of the enzymatic activity. A structure of the C. albicans THI5p shows PLP bound at the active site via an imine with Lys62 and His66 in close proximity to the PLP. Our data suggest that His66 of the THI5 protein is the histidine source for pyrimidine formation and that the pyrimidine synthase is a single-turnover enzyme.
Asunto(s)
Candida albicans/metabolismo , Histidina/metabolismo , Fosfato de Piridoxal/metabolismo , Pirimidinas/biosíntesis , Tiamina/biosíntesis , Candida albicans/química , Histidina/química , Modelos Moleculares , Estructura Molecular , Fosfato de Piridoxal/química , Pirimidinas/química , Tiamina/químicaRESUMEN
Pyruvoyl-dependent arginine decarboxylase (PvlArgDC) catalyzes the first step of the polyamine-biosynthetic pathway in plants and some archaebacteria. The pyruvoyl group of PvlArgDC is generated by an internal autoserinolysis reaction at an absolutely conserved serine residue in the proenzyme, resulting in two polypeptide chains. Based on the native structure of PvlArgDC from Methanococcus jannaschii, the conserved residues Asn47 and Glu109 were proposed to be involved in the decarboxylation and autoprocessing reactions. N47A and E109Q mutant proteins were prepared and the three-dimensional structure of each protein was determined at 2.0 A resolution. The N47A and E109Q mutant proteins showed reduced decarboxylation activity compared with the wild-type PvlArgDC. These residues may also be important for the autoprocessing reaction, which utilizes a mechanism similar to that of the decarboxylation reaction.
Asunto(s)
Carboxiliasas/química , Carboxiliasas/genética , Methanococcus/enzimología , Methanococcus/genética , Sitios de Unión , Carboxiliasas/biosíntesis , Clonación Molecular , Cristalización , Modelos Moleculares , Conformación Molecular , Mutagénesis , Mutación/genética , Piruvatos/metabolismo , Difracción de Rayos XRESUMEN
Thiamin monophosphate kinase (ThiL) catalyzes the ATP-dependent phosphorylation of thiamin monophosphate (TMP) to form thiamin pyrophosphate (TPP), the active form of vitamin B 1. ThiL is a member of a small ATP binding superfamily that also includes the purine biosynthetic enzymes, PurM and PurL, NiFe hydrogenase maturation protein, HypE, and selenophosphate synthase, SelD. The latter four enzymes are believed to utilize phosphorylated intermediates during catalysis. To understand the mechanism of ThiL and its relationship to the other superfamily members, we determined the structure of Aquifex aeolicus ThiL (AaThiL) with nonhydrolyzable AMP-PCP and TMP, and also with the products of the reaction, ADP and TPP. The results suggest that AaThiL utilizes a direct, inline transfer of the gamma-phosphate of ATP to TMP rather than a phosphorylated enzyme intermediate. The structure of ThiL is compared to those of PurM, PurL, and HypE, and the ATP binding site is compared to that of PurL, for which nucleotide complexes are available.
Asunto(s)
Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Bacterias/enzimología , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de SecuenciaRESUMEN
The function of the mlr6793 gene from Mesorhizobium loti MAFF303099 has been identified. This gene encodes 4-pyridoxic acid dehydrogenase, an enzyme involved in the catabolism of PLP (Vitamin B6). This enzyme was overexpressed in Escherichia coli and characterized. 4-Pyridoxic acid dehydrogenase is a 33kDa protein that catalyzes the four electron oxidation of 4-pyridoxic acid to 3-hydroxy-2-methylpyridine-4,5-dicarboxylate, using nicotinamide adenine dinucleotide as a cofactor. The k cat for NADH production is 0.01s(-1). The KM values for 4-pyridoxic acid and NAD are 5.8 and 6.6microM, respectively.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Piridoxina/metabolismo , Rhizobiaceae/enzimología , Cromatografía Líquida de Alta Presión , Clonación Molecular , Escherichia coli/genética , Rhizobiaceae/genéticaRESUMEN
The substrates for Bacillus subtilis PLP synthase (YaaD and YaaE) are identified, and the first reconstitution of PLP biosynthesis using this pathway is described. Three partial reactions catalyzed by YaaD are also identified.
Asunto(s)
Fosfato de Piridoxal/biosíntesis , Antranilato Sintasa/metabolismo , Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Análisis de Fourier , Glutaminasa/metabolismo , Transferasas de Grupos Nitrogenados/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The genes encoding thiamine kinase in Escherichia coli (ycfN) and thiamine pyrophosphokinase in Bacillus subtilis (yloS) have been identified. This study completes the identification of the thiamine salvage enzymes in bacteria.
Asunto(s)
Genes Bacterianos/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Tiamina Pirofosfoquinasa/genética , Tiamina/metabolismo , Bacillus subtilis/genética , Escherichia coli/genética , NAD/metabolismoRESUMEN
S-adenosylmethionine decarboxylase (AdoMetDC) is a critical regulatory enzyme of the polyamine biosynthetic pathway and belongs to a small class of pyruvoyl-dependent amino acid decarboxylases. Structural elucidation of the prokaryotic AdoMetDC is of substantial interest in order to determine the relationship between the eukaryotic and prokaryotic forms of the enzyme. Although both forms utilize pyruvoyl groups, there is no detectable sequence similarity except at the site of pyruvoyl group formation. The x-ray structure of the Thermatoga maritima AdoMetDC proenzyme reveals a dimeric protein fold that is remarkably similar to the eukaryotic AdoMetDC protomer, suggesting an evolutionary link between the two forms of the enzyme. Three key active site residues (Ser55, His68, and Cys83) involved in substrate binding, catalysis or proenzyme processing that were identified in the human and potato AdoMet-DCs are structurally conserved in the T. maritima AdoMetDC despite very limited primary sequence identity. The role of Ser55, His68, and Cys83 in the self-processing reaction was investigated through site-directed mutagenesis. A homology model for the Escherichia coli AdoMetDC was generated based on the structures of the T. maritima and human AdoMetDCs.
Asunto(s)
Adenosilmetionina Descarboxilasa/química , Adenosilmetionina Descarboxilasa/genética , Evolución Molecular , Thermotoga maritima/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Secuencia Conservada , Cristalización , Cristalografía por Rayos X , Dimerización , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Escherichia coli/genética , Expresión Génica , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Pliegue de Proteína , Solanum tuberosum/enzimología , Thermotoga maritima/genéticaRESUMEN
Two Bacillus subtilis genes encoding two proteins (currently annotated ThiD and YjbV) were overexpressed and characterized. YjbV has 4-amino-5-hydroxymethyl-2-methylpyrimidine and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate kinase activity and should be reannotated ThiD, and B. subtilis ThiD has pyridoxine, pyridoxal, and pyridoxamine kinase activity and should be reannotated PdxK.
Asunto(s)
Bacillus subtilis/enzimología , Piridoxal Quinasa/metabolismo , Fosfato de Piridoxal/biosíntesis , Tiamina Pirofosfato/biosíntesis , Pirimidinas/metabolismo , Especificidad por SustratoRESUMEN
Thiamin pyrophosphate was synthesized in 71% yield, on a multi-milligram scale, using overexpressed thiazole kinase, pyrimidine kinase, thiamin phosphate synthase, and thiamin phosphate kinase. This provides a facile route to isotopically labeled thiamin pyrophosphate from its readily available pyrimidine and thiazole precursors.
Asunto(s)
Fosfotransferasas (Aceptor del Grupo Fosfato) , Tiamina Pirofosfato/síntesis química , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Escherichia coli/enzimología , Reacción en Cadena de la Polimerasa , Proteínas RecombinantesRESUMEN
While most of the proteins required for the biosynthesis of thiamin pyrophosphate have been known for more than a decade, the reconstitution of this biosynthesis in a defined biochemical system has been difficult due to the novelty of the chemistry involved. Here we demonstrate the first successful enzymatic synthesis of the thiazole moiety of thiamin from glycine, cysteine, and deoxy-D-xylulose-5-phosphate using overexpressed Bacillus subtilis ThiF, ThiS, ThiO, ThiG, and a NifS-like protein. This has facilitated the identification of the biochemical function of each of the proteins involved: ThiF catalyzes the adenylation of ThiS; NifS catalyzes the transfer of sulfur from cysteine to the acyl adenylate of ThiS; ThiO catalyzes the oxidation of glycine to the corresponding imine; and ThiG catalyzes the formation of the thiazole phosphate ring. The complex oxidative cyclization reaction involved in the biosynthesis of the thiamin thiazole has been greatly simplified by replacing ThiF, ThiS, ThiO, and NifS with defined biosynthetic intermediates in a reaction where ThiG is the only required enzyme.
Asunto(s)
Tiamina/biosíntesis , Bacillus subtilis/genética , Cromatografía en Capa Delgada , Electroforesis en Gel de Poliacrilamida , Cinética , Espectrometría de Masa por Ionización de Electrospray , Tiamina/química , Tiamina/genéticaRESUMEN
Pantothenate kinase (CoaA) catalyzes the first step of the coenzyme A biosynthetic pathway. Here we report the identification of the Staphylococcus aureus coaA gene and characterization of the enzyme. We have also identified a series of low-molecular-weight compounds which are effective inhibitors of S. aureus CoaA.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/enzimología , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Coenzima A/biosíntesis , ADN Bacteriano/química , ADN Bacteriano/genética , Humanos , Concentración 50 Inhibidora , Cinética , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Ácido Pantoténico/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Filogenia , Alineación de Secuencia , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Células Tumorales CultivadasRESUMEN
Oxalate decarboxylase is a manganese-dependent enzyme that catalyzes the conversion of oxalate to formate and carbon dioxide. We have determined the structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution in the presence of formate. The structure reveals a hexamer with 32-point symmetry in which each monomer belongs to the cupin family of proteins. Oxalate decarboxylase is further classified as a bicupin because it contains two cupin folds, possibly resulting from gene duplication. Each oxalate decarboxylase cupin domain contains one manganese binding site. Each of the oxalate decarboxylase domains is structurally similar to oxalate oxidase, which catalyzes the manganese-dependent oxidative decarboxylation of oxalate to carbon dioxide and hydrogen peroxide. Amino acid side chains in the two metal binding sites of oxalate decarboxylase and the metal binding site of oxalate oxidase are very similar. Four manganese binding residues (three histidines and one glutamate) are conserved as well as a number of hydrophobic residues. The most notable difference is the presence of Glu333 in the metal binding site of the second cupin domain of oxalate decarboxylase. We postulate that this domain is responsible for the decarboxylase activity and that Glu333 serves as a proton donor in the production of formate. Mutation of Glu333 to alanine reduces the catalytic activity by a factor of 25. The function of the other domain in oxalate decarboxylase is not yet known.