RESUMEN

Melatonin (Mel) has previously been reported to effectively alleviate nitrogen-limitation (N-L) stress and thus increase nitrogen-use efficiency (NUE) in several plants, but the underlying mechanism remains obscure. Here, we revealed that OsbZIP79 (BASIC LEUCINE ZIPPER 79) is transcriptionally activated under N-L conditions, and its expression is further enhanced by exogenous Mel. By the combined use of omics, genetics, and biological techniques, we revealed that the OsbZIP79-OsABI5 (ABSCISIC ACID INSENSITIVE 5) module stimulated regulation of reactive oxygen species (ROS) homeostasis and the uptake and metabolism of nitrogen under conditions of indoor nitrogen limitation (1/16 normal level). OsbZIP79 activated the transcription of OsABI5, and OsABI5 then bound to the promoters of target genes, including genes involved in ROS homeostasis and nitrogen metabolism, activating their transcription. This module was also indispensable for upregulation of several other genes involved in abscisic acid catabolism, nitrogen uptake, and assimilation under N-L and Mel treatment, although these genes were not directly transactivated by OsABI5. Field experiments demonstrated that Mel significantly improved rice growth under low nitrogen (L-N, half the normal level) by the same mechanism revealed in the nitrogen-limitation study. Mel application produced a 28.6% yield increase under L-N and thus similar increases in NUE. Also, two OsbZIP79-overexpression lines grown in L-N field plots had significantly higher NUE (+13.7% and +21.2%) than their wild types. Together, our data show that an OsbZIP79-OsABI5 module regulates the rice response to N insufficiency (N limitation or low N), which is important for increasing NUE in rice production.

Asunto(s)

Melatonina , Oryza , Melatonina/farmacología , Melatonina/metabolismo , Ácido Abscísico/metabolismo , Oryza/genética , Oryza/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Nitrógeno/metabolismo , Homeostasis/genética

RESUMEN

Allantoinase (ALLase; EC 3.5.2.5) possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carbamylated lysine. ALLase acts as a key enzyme for the biogenesis and degradation of ureides by catalyzing the conversion of allantoin into allantoate. Biochemically, ALLase belongs to the cyclic amidohydrolase family, which also includes dihydropyrimidinase, dihydroorotase, hydantoinase (HYDase), and imidase. Previously, the crystal structure of ALLase from Escherichia coli K-12 (EcALLase-K12) was reported; however, the two active site loops crucial for substrate binding were not determined. This situation would limit further docking and protein engineering experiments. Here, we solved the crystal structure of E. coli BL21 ALLase (EcALLase-BL21) at a resolution of 2.07 Å (PDB ID 8HFD) to obtain more information for structural analyses. The structure has a classic TIM barrel fold. As compared with the previous work, the two missed active site loops in EcALLase-K12 were clearly determined in our structure of EcALLase-BL21. EcALLase-BL21 shared active site similarity with HYDase, an important biocatalyst for industrial production of semisynthetic penicillin and cephalosporins. Based on this structural comparison, we discussed the functional role of the two active site loops in EcALLase-BL21 to better understand the substrate/inhibitor binding mechanism for further biotechnological and pharmaceutical applications.

Asunto(s)

Escherichia coli K12 , Escherichia coli , Escherichia coli/metabolismo , Dominio Catalítico , Amidohidrolasas/química , Catálisis , Cristalografía por Rayos X , Sitios de Unión

RESUMEN

Nepenthes are carnivorous pitcher plants that have several ethnobotanical uses, such as curing stomachache and fever. Here, we prepared different extracts from the stem, leaf, and pitcher of Nepenthes miranda to further investigate their pharmacological potential. The leaf extract of N. miranda obtained by 100% acetone (N. miranda-leaf-acetone) was used in this study to analyze the cytotoxic activities, antioxidation capacity, antibacterial activity, and allantoinase (ALLase) inhibitory effect of this plant. The cytotoxic effects of N. miranda-leaf-acetone on the survival, apoptosis, and migration of the cancer cell lines PC-9 pulmonary adenocarcinoma, B16F10 melanoma, and 4T1 mammary carcinoma cells were demonstrated. Based on collective data, the cytotoxic activities of N. miranda-leaf-acetone followed the order: B16F10 > 4T1 > PC-9 cells. In addition, the cytotoxic activities of N. miranda-leaf-acetone were synergistically enhanced when co-acting with the clinical anticancer drug 5-fluorouracil. N. miranda-leaf-acetone could also inhibit the activity of ALLase, a key enzyme in the catabolism pathway for purine degradation. Through gas chromatography−mass spectrometry, the 16 most abundant ingredients in N. miranda-leaf-acetone were identified. The top six compounds in N. miranda-leaf-acetone, namely, plumbagin, lupenone, palmitic acid, stigmast-5-en-3-ol, neophytadiene, and citraconic anhydride, were docked to ALLase, and their docking scores were compared. The docking results suggested plumbagin and stigmast-5-en-3-ol as potential inhibitors of ALLase. Overall, these results may indicate the pharmacological potential of N. miranda for further medical applications.

RESUMEN

Dihydroorotase (DHOase), a dimetalloenzyme containing a carbamylated lysine within the active site, is a member of the cyclic amidohydrolase family, which also includes allantoinase (ALLase), dihydropyrimidinase (DHPase), hydantoinase, and imidase. Unlike most known cyclic amidohydrolases, which are tetrameric, DHOase exists as a monomer or dimer. Here, we report and analyze two crystal structures of the eukaryotic Saccharomyces cerevisiae DHOase (ScDHOase) complexed with malate. The structures of different DHOases were also compared. An asymmetric unit of these crystals contained four crystallographically independent ScDHOase monomers. ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). Unlike EcDHOase, ScDHOase can form tetramers, both in the crystalline state and in solution. In addition, the subunit-interacting residues of ScDHOase for dimerization and tetramerization are significantly different from those of other DHOases. The tetramerization pattern of ScDHOase is also different from those of DHPase and ALLase. Based on sequence analysis and structural evidence, we identify two unique helices (α6 and α10) and a loop (loop 7) for tetramerization, and discuss why the residues for tetramerization in ScDHOase are not necessarily conserved among DHOases.

Asunto(s)

Dihidroorotasa/química , Dihidroorotasa/metabolismo , Multimerización de Proteína , Saccharomyces cerevisiae/enzimología , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Biocatálisis , Cristalografía por Rayos X , Estabilidad de Enzimas , Humanos , Enlace de Hidrógeno , Lisina/metabolismo , Malatos/metabolismo , Modelos Moleculares , Saccharomyces cerevisiae/genética , Soluciones , Temperatura

RESUMEN

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides, and an attractive target for potential anticancer chemotherapy. By screening plant extracts and performing GC-MS analysis, we identified and characterized that the potent anticancer drug plumbagin (PLU), isolated from the carnivorous plant Nepenthes miranda, was a competitive inhibitor of DHOase. We also solved the complexed crystal structure of yeast DHOase with PLU (PDB entry 7CA1), to determine the binding interactions and investigate the binding modes. Mutational and structural analyses indicated the binding of PLU to DHOase through loop-in mode, and this dynamic loop may serve as a drug target. PLU exhibited cytotoxicity on the survival, migration, and proliferation of 4T1 cells and induced apoptosis. These results provide structural insights that may facilitate the development of new inhibitors targeting DHOase, for further clinical anticancer chemotherapies.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Productos Biológicos/farmacología , Vías Biosintéticas/efectos de los fármacos , Dihidroorotasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Naftoquinonas/farmacología , Pirimidinas/biosíntesis , Antineoplásicos Fitogénicos/química , Sitios de Unión , Productos Biológicos/química , Dominio Catalítico , Dihidroorotasa/química , Dihidroorotasa/genética , Inhibidores Enzimáticos/química , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Mutación , Naftoquinonas/química , Unión Proteica , Relación Estructura-Actividad

RESUMEN

Dihydropyrimidinase catalyzes the reversible hydrolytic ring opening of dihydrouracil and dihydrothymine to N-carbamoyl-ß-alanine and N-carbamyl-ß-aminoisobutyrate, respectively. Dihydropyrimidinase from microorganisms is normally known as hydantoinase because of its role as a biocatalyst in the synthesis of d- and l-amino acids for the industrial production of antibiotic precursors and its broad substrate specificity. Dihydropyrimidinase belongs to the cyclic amidohydrolase family, which also includes imidase, allantoinase, and dihydroorotase. Although these metal-dependent enzymes share low levels of amino acid sequence homology, they possess similar active site architectures and may use a similar mechanism for catalysis. By contrast, the five human dihydropyrimidinase-related proteins possess high amino acid sequence identity and are structurally homologous to dihydropyrimidinase, but they are neuronal proteins with no dihydropyrimidinase activity. In this chapter, we summarize and discuss current knowledge and the recent advances on the structure, catalytic mechanism, and inhibition of dihydropyrimidinase.

Asunto(s)

Amidohidrolasas/química , Lisina/química , Carbamilación de Proteína , Amidohidrolasas/metabolismo , Lisina/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Homología Estructural de Proteína , Especificidad por Sustrato

RESUMEN

Plants can respond and adapt to changes in the internal content of carbon and nitrogen by using organic compounds that widely differ in their carbon/nitrogen ratio. Among them, the amides asparagine and glutamine are believed to be preferred by most plants, including Arabidopsis. However, increases in the ureides allantoin and/or allantoate concentrations have been observed in different plant species under several environmental conditions. In this work, changes in the ratio between carbon skeletons and reduced nitrogen were investigated by varying the concentrations of nitrogen and sucrose in the growth media. Allantoin accumulation was observed when plants were grown in media with high ammonia concentrations. This increase was reverted by adding sucrose as additional carbon source. Moreover, mutant plants with a decreased capability to degrade allantoin showed a compromised growth compared to WT in ammonia supplemented media. Together, our results indicate that allantoin accumulation is induced by low carbon/nitrogen ratio and suggest that its degradation is critical for proper plant growth and development.

Asunto(s)

Alantoína , Proteínas de Arabidopsis , Arabidopsis , Alantoína/metabolismo , Amidohidrolasas , Arabidopsis/genética , Arabidopsis/metabolismo , Nitrógeno , Hojas de la Planta

RESUMEN

Dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. This enzyme is important in pyrimidine metabolism, and blocking its activity would be detrimental to cell survival. This study investigated the dihydropyrimidinase inhibition by plumbagin isolated from the extract of carnivorous plant Nepenthes miranda (Nm). Plumbagin inhibited dihydropyrimidinase with IC50 value of 58 ± 3 µM. Double reciprocal results of Lineweaver-Burk plot indicated that this compound is a competitive inhibitor of dihydropyrimidinase. Fluorescence quenching analysis revealed that plumbagin could form a stable complex with dihydropyrimidinase with the Kd value of 37.7 ± 1.4 µM. Docking experiments revealed that the dynamic loop crucial for stabilization of the intermediate state in dihydropyrimidinase might be involved in the inhibition effect of plumbagin. Mutation at either Y155 or K156 within the dynamic loop of dihydropyrimidinase caused low plumbagin binding affinity. In addition to their dihydropyrimidinase inhibition, plumbagin and Nm extracts also exhibited cytotoxicity on melanoma cell survival, migration, and proliferation. Further research can directly focus on designing compounds that target the dynamic loop in dihydropyrimidinase during catalysis.

Asunto(s)

Amidohidrolasas/antagonistas & inhibidores , Bacterias/efectos de los fármacos , Magnoliopsida/química , Naftoquinonas/farmacología , Extractos Vegetales/farmacología , Animales , Antibacterianos/farmacología , Antineoplásicos/farmacología , Antioxidantes/farmacología , Línea Celular , Línea Celular Tumoral , Ratones

RESUMEN

Ureides are nitrogenous compounds with a special function in some legume under nitrogen fixing conditions, the ureidic legumes. In this group, ureides are the predominant nitrogen transport molecule from nodules to the upper part, whereas amidic legumes use amides as nitrogen transport compounds. In this study, the ureide levels have been analysed in seedlings from four ureidic and four amidic legume plants. It has been found that the differentiation among ureide and amide plants already exists in seedlings during early seedling development, with high levels of ureide and allantoinase activity in cotyledons and embryonic axes from ureide plants. Since ureides have been implicated in the response of plant to several stress, total hydrosoluble antioxidant capacity and the levels of several antioxidant activities have been determined and compared among these two legume groups. The total antioxidant capacity did not follow any differential pattern in cotyledons or embryonic axes for the analysed plants. The levels of superoxide dismutase, guaiacol peroxidase and ascorbate peroxidase in both embryonic axes and cotyledons are statistical different between amide and ureide seedlings, whereas the catalase activity was similar among these groups of plants. We discuss than amides and ureides could follow different strategies to protect against oxidation.

Asunto(s)

Antioxidantes/metabolismo , Cotiledón/metabolismo , Fabaceae/metabolismo , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Plantones/metabolismo , Fijación del Nitrógeno/fisiología

RESUMEN

Allantoin, the natural end product of purine catabolism in mammals, is non-enzymatically produced from the scavenging of reactive oxygen species through the degradation of uric acid. Levels of allantoin in biological fluids are sensitively influenced by the presence of free radicals, making this molecule a candidate marker of acute oxidative stress in clinical analyses. With this aim, we exploited allantoinase-the enzyme responsible for allantoin hydrolization in plants and lower organisms-for the development of a biosensor exploiting a fast enzymatic-chemical assay for allantoin quantification. Recombinant allantoinase was entrapped in a wet nanoporous silica gel matrix and its structural properties, function, and stability were characterized through fluorescence spectroscopy and circular dichroism measurements, and compared to the soluble enzyme. Physical immobilization in silica gel minimally influences the structure and the catalytic efficiency of entrapped allantoinase, which can be reused several times and stored for several months with good activity retention. These results, together with the relative ease of the sol-gel preparation and handling, make the encapsulated allantoinase a good candidate for the development of an allantoin biosensor.

Asunto(s)

Amidohidrolasas/metabolismo , Técnicas Biosensibles , Enzimas Inmovilizadas/metabolismo , Fenómenos Ópticos , Estrés Oxidativo , Amidohidrolasas/química , Biocatálisis , Enzimas Inmovilizadas/química , Geles/química , Cinética , Conformación Proteica , Dióxido de Silicio/química , Cloruro de Sodio/farmacología , Factores de Tiempo

RESUMEN

Dihydropyrimidinase (DHPase) is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase (DHOase), hydantoinase, and imidase. Almost all of these zinc metalloenzymes possess a binuclear metal center in which two metal ions are bridged by a post-translational carbamylated Lys. Crystal structure of Tetraodon nigroviridis DHPase reveals that one zinc ion is sufficient to stabilize Lys carbamylation. In this study, we found that one metal coordination was not sufficient to fix CO2 to the Lys in bacterial DHPase. We prepared and characterized mono-Zn DHPase from Pseudomonas aeruginosa (PaDHPase), and the catalytic activity of mono-Zn PaDHPase was not detected. The crystal structure of mono-Zn PaDHPase determined at 2.23 Šresolution (PDB entry 6AJD) revealed that Lys150 was no longer carbamylated. This finding indicated the decarbamylation of the Lys during the metal chelating process. To confirm the state of Lys carbamylation in mono-Zn PaDHPase in solution, mass spectrometric (MS) analysis was carried out. The MS result was in agreement with the theoretical value for uncarbamylated PaDHPase. Crystal structure of the human DHOase domain (huDHOase) K1556A mutant was also determined (PDB entry 5YNZ), and the structure revealed that the active site of huDHOase K1556A mutant contained one metal ion. Like mono-Zn PaDHPase, oxygen ligands of the carbamylated Lys were not required for Znα binding. Considering the collective data from X-ray crystal structure and MS analysis, mono-Zn PaDHPase in both crystalline state and solution was not carbamylated. In addition, structural evidences indicated that post-translational carbamylated Lys was not required for Znα binding in PaDHPase and in huDHOase.

Asunto(s)

Amidohidrolasas/química , Dihidroorotasa/química , Lisina/metabolismo , Carbamilación de Proteína , Proteínas Bacterianas/química , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Espectrometría de Masas , Proteínas Mutantes/química , Unión Proteica , Procesamiento Proteico-Postraduccional , Pseudomonas aeruginosa/enzimología , Zinc/metabolismo

RESUMEN

Dihydropyrimidinase, a tetrameric metalloenzyme, is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. In this paper, we report the crystal structure of dihydropyrimidinase from Pseudomonas aeruginosa PAO1 at 2.1 Å resolution. The structure of P. aeruginosa dihydropyrimidinase reveals a classic (ß/α)8-barrel structure core embedding the catalytic dimetal center and a ß-sandwich domain, which is commonly found in the architecture of dihydropyrimidinases. In contrast to all dihydropyrimidinases, P. aeruginosa dihydropyrimidinase forms a dimer, rather than a tetramer, both in the crystalline state and in the solution. Basing on sequence analysis and structural comparison of the C-terminal region and the dimer-dimer interface between P. aeruginosa dihydropyrimidinase and Thermus sp. dihydropyrimidinase, we propose a working model to explain why this enzyme cannot be a tetramer.

Asunto(s)

Amidohidrolasas/química , Pseudomonas aeruginosa/enzimología , Secuencia de Aminoácidos , Cromatografía en Gel , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Multimerización de Proteína , Sales (Química)/química , Soluciones , Thermus/enzimología

RESUMEN

Allantoin, a metabolite generated in the purine degradation pathway, was primarily considered an intermediate for recycling of the abundant nitrogen assimilated in plant purines. More specifically, tropical legumes utilize allantoin and allantoic acid as major nodule-to-shoot nitrogen transport compounds. In other species, an increase in allantoin content was observed under different stress conditions, but the underlying molecular mechanisms remain poorly understood. In this work, Arabidopsis thaliana was used as a model system to investigate the effects of salt stress on allantoin metabolism and to know whether its accumulation results in plant protection. Plant seedlings treated with NaCl at different concentrations showed higher allantoin and lower allantoic acid contents. Treatments with NaCl favored the expression of genes involved in allantoin synthesis, but strongly repressed the unique gene encoding allantoinase (AtALN). Due to the potential regulatory role of this gene for allantoin accumulation, AtALN promoter activity was studied using a reporter system. GUS mediated coloration was found in specific plant tissues and was diminished with increasing salt concentrations. Phenotypic analysis of knockout, knockdown and stress-inducible mutants for AtALN revealed that allantoin accumulation is essential for salt stress tolerance. In addition, the possible role of allantoin transport was investigated. The Ureide Permease 5 (UPS5) is expressed in the cortex and endodermis of roots and its transcription is enhanced by salt treatment. Ups5 knockout plants under salt stress presented a susceptible phenotype and altered allantoin root-to-shoot content ratios. Possible roles of allantoin as a protectant compound in oxidative events or signaling are discussed.

Asunto(s)

Alantoína/metabolismo , Amidohidrolasas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/fisiología , Regulación hacia Abajo , Proteínas de Transporte de Membrana/metabolismo , Tolerancia a la Sal , Estrés Fisiológico , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico/efectos de los fármacos , ADN Bacteriano/genética , Regulación hacia Abajo/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Técnicas de Inactivación de Genes , Genes de Plantas , Proteínas de Transporte de Membrana/genética , Mutagénesis Insercional/genética , Fenotipo , Desarrollo de la Planta/efectos de los fármacos , Desarrollo de la Planta/genética , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Tolerancia a la Sal/efectos de los fármacos , Plantones/efectos de los fármacos , Plantones/metabolismo , Cloruro de Sodio/farmacología , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Transcripción Genética/efectos de los fármacos

RESUMEN

Allantoinase, a member of the amidohydrolase superfamily, exists in a wide variety of organisms, including bacteria, fungi, plants and a few animals, such as fishes and amphibians. Allantoinase catalyzes the reversible hydrolysis of allantoin into allantoate by hydrolytic cleavage of the N1-C2 amide bond of the five-membered hydantoin ring. Allantoinase from Bacillus licheniformis (AllBali) presents an inverted enantioselectivity towards allantoin (R-enantioselective), which is a distinguishable feature that is not observed for other allantoinases. In this work, B. licheniformis ATCC 14580 allantoinase (AllBali) containing a C-terminal His6 tag was overproduced in Escherichia coli and purified to homogeneity. Crystals of AllBali were obtained by the vapour-diffusion method using 0.1 M potassium thiocyanate, 20%(w/v) polyethylene glycol 3350 as a crystallization solution. X-ray diffraction data were collected to a resolution of 3.5 Šwith an Rmerge of 29.2% from a crystal belonging to space group P1211, with unit-cell parameters a=54.93, b=164.74, c=106.89 Å, ß=98.49°. There are four molecules in the asymmetric unit with a solvent content of 47% as estimated from the Matthews coefficient (VM=2.34 Å3 Da(-1)).

Asunto(s)

Amidohidrolasas/biosíntesis , Amidohidrolasas/química , Bacillus/enzimología , Clonación Molecular , Regulación Bacteriana de la Expresión Génica , Amidohidrolasas/aislamiento & purificación , Clonación Molecular/métodos , Difracción de Rayos X

RESUMEN

Allantoinase and dihydroorotase are members of the cyclic amidohydrolases family. Allantoinase and dihydroorotase possess very similar binuclear metal centers in the active site and may use a similar mechanism for catalysis. However, whether the substrate specificities of allantoinase and dihydroorotase overlap and whether the substrates of other cyclic amidohydrolases inhibit allantoinase and dihydroorotase remain unknown. In this study, the binding and inhibition of allantoinase (Salmonella enterica serovar Typhimurium LT2) and dihydroorotase (Klebsiella pneumoniae) by flavonols and the substrates of other cyclic amidohydrolases were investigated. Hydantoin and phthalimide, substrates of hydantoinase and imidase, were not hydrolyzed by allantoinase and dihydroorotase. Hydantoin and dihydroorotate competitively inhibited allantoinase, whereas hydantoin and allantoin bind to dihydroorotase, but do not affect its activity. We further investigated the effects of the flavonols myricetin, quercetin, kaempferol, and galangin, on the inhibition of allantoinase and dihydroorotase. Allantoinase and dihydroorotase were both significantly inhibited by kaempferol, with IC50 values of 35 ± 3 µM and 31 ± 2 µM, respectively. Myricetin strongly inhibited dihydroorotase, with an IC50 of 40 ± 1 µM. The double reciprocal of the Lineweaver-Burk plot indicated that kaempferol was a competitive inhibitor for allantoinase but an uncompetitive inhibitor for dihydroorotase. A structural study using PatchDock showed that kaempferol was docked in the active site pocket of allantoinase but outside the active site pocket of dihydroorotase. These results constituted a first study that naturally occurring product flavonols inhibit the cyclic amidohydrolases, allantoinase, and dihydroorotase, even more than the substrate analogs (>3 orders of magnitude). Thus, flavonols may serve as drug leads for designing compounds that target several cyclic amidohydrolases.

Asunto(s)

Amidohidrolasas/química , Proteínas Bacterianas/química , Dihidroorotasa/química , Inhibidores Enzimáticos/química , Quempferoles/química , Amidohidrolasas/antagonistas & inhibidores , Proteínas Bacterianas/antagonistas & inhibidores , Unión Competitiva , Dominio Catalítico , Dihidroorotasa/antagonistas & inhibidores , Hidantoínas/química , Klebsiella pneumoniae/enzimología , Modelos Moleculares , Ácido Orótico/análogos & derivados , Ácido Orótico/química , Ftalimidas/química , Unión Proteica , Salmonella typhimurium/enzimología , Especificidad por Sustrato

RESUMEN

Allantoinases (allantoin amidohydrolase, E.C. 3.5.2.5) catalyze the hydrolysis of the amide bond of allantoin to form allantoic acid, in those organisms where allantoin is not the final product of uric acid degradation. Despite their importance in the purine catabolic pathway, sequences of microbial allantoinases with proven activity are scarce, and only the enzyme from Escherichia coli (AllEco) has been studied in detail in the genomic era. In this work, we report the cloning, purification and characterization of the recombinant allantoinase from Bacillus licheniformis CECT 20T (AllBali). The enzyme was a homotetramer with an apparent Tm of 62 ± 1 °C. Optimal parameters for the enzyme activity were pH 7.5 and 50 °C, showing apparent Km and kcat values of 17.7 ± 2.7 mM and 24.4 ± 1.5 s(-1), respectively. Co(2+) proved to be the most effective cofactor, inverting the enantioselectivity of AllBali when compared to that previously reported for other allantoinases. The common ability of different cyclic amidohydrolases to hydrolyze distinct substrates to the natural one also proved true for AllBali. The enzyme was able to hydrolyze hydantoin, dihydrouracil and 5-ethyl-hydantoin, although at relative rates 3-4 orders of magnitude lower than with allantoin. Mutagenesis experiments suggest that S292 is likely implicated in the binding of the allantoin ring through the carbonyl group of the polypeptide main chain, which is the common mechanism observed in other members of the amidohydrolase family. In addition, our results suggest an allosteric effect of H2O2 toward allantoinase.

Asunto(s)

Amidohidrolasas/química , Bacillus/enzimología , Proteínas Bacterianas/química , Alantoína/química , Regulación Alostérica , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/genética , Sustitución de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Dominio Catalítico , Cobalto/química , Cisteína/química , Inhibidores Enzimáticos/química , Hidantoínas/química , Peróxido de Hidrógeno/química , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Manganeso/química , Peso Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estereoisomerismo , Especificidad por Sustrato , Temperatura de Transición , Uracilo/análogos & derivados , Uracilo/química

RESUMEN

The end product of purine catabolism varies amongst vertebrates and is a consequence of independent gene inactivation events that have truncated the purine catabolic pathway. Mammals have traditionally been grouped into two classes based on their end product of purine catabolism: most mammals, whose end product is allantoin due to an ancient loss of allantoinase (ALLN), and the hominoids, whose end product is uric acid due to recent inactivations of urate oxidase (UOX). However little is known about purine catabolism in marsupials and monotremes. Here we report the results of a comparative genomics study designed to characterize the purine catabolic pathway in a marsupial, the South American opossum (Monodelphis domestica), and a monotreme, the platypus (Ornithorhynchus anatinus). We found that both genomes encode a more complete set of genes for purine catabolism than do eutherians and conclude that a near complete purine catabolic pathway was present in the common ancestor of all mammals, and that the loss of ALLN is specific to placental mammals. Our results therefore provide a revised history for gene loss in the purine catabolic pathway and suggest that marsupials and monotremes represent a third class of mammals with respect to their end products of purine catabolism.