RESUMEN
Recently, the zinc metallo-hydrolase family of the beta-lactamase fold has grown quite rapidly, accompanied by the accumulation of sequence and structure data. The variety of the biological functions of the family is higher than expected. In addition, the members often have mosaic structures with additional domains. The family includes class B beta-lactamase, glyoxalase II, arylsulfatase, flavoprotein, cyclase/dehydrase, an mRNA 3'-processing protein, a DNA cross-link repair enzyme, a DNA uptake-related protein, an alkylphosphonate uptake-related protein, CMP-N-acetylneuraminate hydroxylase, the romA gene product, alkylsulfatase, and insecticide hydrolases. In this minireview, the functional and structural varieties of the growing protein family are described.
Asunto(s)
beta-Lactamasas/química , Secuencia de Aminoácidos , Animales , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are accessory proteins essential for processive DNA synthesis in the domain Eucarya. The function of RFC is to load PCNA, a processivity factor of eukaryotic DNA polymerases delta and epsilon, onto primed DNA templates. RFC-like genes, arranged in tandem in the Pyrococcus furiosus genome, were cloned and expressed individually in Escherichia coli cells to determine their roles in DNA synthesis. The P. furiosus RFC (PfuRFC) consists of a small subunit (RFCS) and a large subunit (RFCL). Highly purified RFCS possesses an ATPase activity, which was stimulated up to twofold in the presence of both single-stranded DNA (ssDNA) and P. furiosus PCNA (PfuPCNA). The ATPase activity of PfuRFC itself was as strong as that of RFCS. However, in the presence of PfuPCNA and ssDNA, PfuRFC exhibited a 10-fold increase in ATPase activity under the same conditions. RFCL formed very large complexes by itself and had an extremely weak ATPase activity, which was not stimulated by PfuPCNA and DNA. The PfuRFC stimulated PfuPCNA-dependent DNA synthesis by both polymerase I and polymerase II from P. furiosus. We propose that PfuRFC is required for efficient loading of PfuPCNA and that the role of RFC in processive DNA synthesis is conserved in Archaea and Eucarya.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio , Proteínas/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatasas/metabolismo , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/metabolismo , Clonación Molecular , ADN Polimerasa I/metabolismo , ADN Polimerasa II/metabolismo , Replicación del ADN , ADN de Archaea/metabolismo , Genes Arqueales , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Filogenia , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas/metabolismo , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Replicación C , TemperaturaRESUMEN
Animal myeloperoxidase and its relatives constitute a diverse protein family, which includes myeloperoxidase, eosinophil peroxidase, thyroid peroxidase, salivary peroxidase, lactoperoxidase, ovoperoxidase, peroxidasin, peroxinectin, cyclooxygenase, and others. The members of this protein family share a catalytic domain of about 500 amino acid residues in length, although some members have distinctive mosaic structures. To investigate the evolution of the protein family, we performed a comparative analysis of its members, using the amino acid sequences and the coordinate data available today. The results obtained in this study are as follows: (1) 60 amino acid sequences belonging to this family were collected by database searching. We found a new member of the myeloperoxidase family derived from a bacterium. This is the first report of a bacterial member of this family. (2) An unrooted phylogenetic tree of the family was constructed according to the alignment. Considering the branching pattern in the obtained phylogenetic tree, together with the mosaic features in the primary structures, 60 members of the myeloperoxidase family were classified into 16 subfamilies. (3) We found two molecular features that distinguish cyclooxygenase from the other members of the protein family. (4) Several structurally deviated segments were identified by a structural comparison between cyclooxygenase and myeloperoxidase. Some of the segments seemed to be associated with the functional and/or structural differences between the enzymes.
Asunto(s)
Evolución Molecular , Peroxidasa/genética , Secuencia de Aminoácidos , Animales , Bases de Datos Factuales , Humanos , Datos de Secuencia Molecular , Peroxidasa/clasificación , Filogenia , Prostaglandina-Endoperóxido Sintasas/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Hjc resolvase is an archaeal enzyme involved in homologous DNA recombination at the Holliday junction intermediate. However, the structure and the catalytic mechanism of the enzyme have not yet been identified. We performed database searching using the amino acid sequence of the enzyme from Pyrococcus furiosus as a query. We detected 59 amino acid sequences showing weak but significant sequence similarity to the Hjc resolvase. The detected sequences included DPN:II, HAE:II and Vsr endonuclease, which belong to the type II restriction endonuclease family. In addition, a highly conserved region was identified from a multiple alignment of the detected sequences, which was similar to an active site of the type II restriction endonucleases. We substituted three conserved amino acid residues in the highly conserved region of the Hjc resolvase with Ala residues. The amino acid replacements inactivated the enzyme. The experimental study, together with the results of the database searching, suggests that the Hjc resolvase is a distantly related member of the type II restriction endonuclease family. In addition, the results of our database searches suggested that the members of the RecB domain superfamily are evolutionarily related to the type II restriction endonuclease family.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo II/genética , Endodesoxirribonucleasas/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Arqueales/genética , Bases de Datos Factuales , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Endodesoxirribonucleasas/metabolismo , Resolvasas de Unión Holliday , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Pyrococcus furiosus/enzimología , Pyrococcus furiosus/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The Holliday junction cleavage protein, Hjc resolvase of Pyrococcus furiosus, is the first Holliday junction resolvase to be discovered in Archaea. Although the archaeal resolvase shares certain biochemical properties with other non-archaeal junction resolvases, no amino acid sequence similarity has been identified. To investigate the structure-function relationship of this new Holliday junction resolvase, we constructed a series of mutant hjc genes using site-directed mutagenesis targeted at the residues conserved among the archaeal orthologs. The products of these mutant genes were purified to homogeneity. With analysis of the activity of the mutant proteins to bind and cleave synthetic Holliday junctions, one acidic residue, Glu-9, and two basic residues, Arg-10 and Arg-25, were found to play critical roles in enzyme action. This is in addition to the three conserved residues, Asp-33, Glu-46, and Lys-48, which are also conserved in the motif found in the type II restriction endonuclease family proteins. Two aromatic residues, Phe-68 and Phe-72, are important for the formation of the homodimer probably through hydrophobic interactions. The results of these studies have provided insights into the structure-function relationships of the archaeal Holliday junction resolvase as well as the universality and diversity of the Holliday junction cleavage reaction.
Asunto(s)
ADN Bacteriano/metabolismo , Endodesoxirribonucleasas/metabolismo , Pyrococcus furiosus/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , ADN Bacteriano/química , Dimerización , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Resolvasas de Unión Holliday , Hidrólisis , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Unión Proteica , Pyrococcus furiosus/genética , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Cryptochromes (CRY), members of the DNA photolyase/cryptochrome protein family, regulate the circadian clock in animals and plants. Two types of animal CRYs are known, mammalian CRY and Drosophila CRY. Both CRYs participate in the regulation of circadian rhythm, but they have different light dependencies for their reactions and have different effects on the negative feedback loop which generates a circadian oscillation of gene expression. Mammalian CRYs act as a potent inhibitor of transcriptional activator whose reactions do not depend on light, but Drosophila CRY functions as a light-dependent suppressor of transcriptional inhibitor. RESULTS: We cloned seven zebrafish genes that carry members of the DNA photolyase/cryptochrome protein family; one (6-4)photolyase and six cry genes. A sequence analysis and determination of their in vitro functions showed that these zebrafish cry genes constitute two groups. One has a high sequence similarity to mammalian cry genes and inhibits CLOCK:BMAL1 mediated transcription. The other, which has a higher sequence similarity to the Drosophila cry gene rather than the mammalian cry genes, does not carry transcription inhibitor activity. The expressions of these cry genes oscillate in a circadian manner, but their patterns differ. CONCLUSIONS: These findings suggest that functionally diverse cry genes are present in zebrafish and each gene has different role in the molecular clock.
Asunto(s)
Ritmo Circadiano , Desoxirribodipirimidina Fotoliasa/genética , Proteínas de Drosophila , Proteínas del Ojo , Flavoproteínas/genética , Células Fotorreceptoras de Invertebrados , Pez Cebra/genética , Factores de Transcripción ARNTL , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Relojes Biológicos , Northern Blotting , Proteínas CLOCK , Secuencia Conservada , Criptocromos , Desoxirribodipirimidina Fotoliasa/metabolismo , Evolución Molecular , Flavoproteínas/metabolismo , Datos de Secuencia Molecular , Fotoperiodo , Filogenia , Receptores Acoplados a Proteínas G , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Transactivadores/antagonistas & inhibidores , Transactivadores/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Rayos Ultravioleta , Pez Cebra/metabolismoRESUMEN
Archaeal RadA, like eukaryotic Rad51 and bacterial RecA, promotes strand exchange between DNA strands with homologous sequences in vitro and is believed to participate in the homologous recombination in cells. The amino acid sequences of the archaeal RadA proteins are more similar to the eukaryotic Rad51s rather than the bacterial RecAs, and the N-terminal region containing domain I is conserved among the RadA and Rad51 proteins but is absent from RecA. To understand the structure-function relationship of RadA, we divided the RadA protein from Pyrococcus furiosus into two parts, the N-terminal one-third (RadA-n) and the residual C-terminal two-thirds (RadA-c), the latter of which contains the central core domain (domain II) of the RecA/Rad51 family proteins. RadA-c had the DNA-dependent ATPase activity and the strand exchange activity by itself, although much weaker (10%) than that of the intact RadA. These activities of RadA-c were restored to 60% of those of RadA by addition of RadA-n, indicating that the proper active structure of RadA was reconstituted in vitro. These results suggest that the basic activities of the RecA/Rad51 family proteins for homologous recombination are derived from domain II, and the N-terminal region may help to enhance the catalytic efficiencies.
Asunto(s)
Proteínas Arqueales , Proteínas de Unión al ADN/química , Pyrococcus furiosus/genética , Recombinación Genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , ADN/metabolismo , Datos de Secuencia Molecular , Recombinasa Rad51 , Relación Estructura-ActividadRESUMEN
Photolyase is a DNA repair enzyme that reverses UV-induced photoproducts in DNA in a light-dependent manner. Recently, photolyase homologs were identified in higher eukaryotes. These homologs, termed crypto-chromes, function as blue light photoreceptors or regulators of circadian rhythm. In contrast, most bacteria have only a single photolyase or photolyase-like gene. Unlike other microbes, the chromosome of the cyanobacterium SYNECHOCYSTIS: sp. PCC6803 contains two ORFs (slr0854 and sll1629) with high similarities to photolyases. We have characterized both genes. The slr0854 gene product exhibited specific, light-dependent repair activity for a cyclo-butane pyrimidine dimer (CPD), whereas the sll1629 gene product lacks measurable affinity for DNA in vitro. Disruption of either slr0854 or sll1629 had little or no effect on the growth rate of the cyanobacterium. A mutant lacking the slr0854 gene showed severe UV sensitivity, in contrast to a mutant lacking sll1629. Phylogenetic analysis showed that sll1629 is more closely related to the cryptochromes than photolyases. We conclude that sll1629 is a bacterial cryptochrome. To our knowledge, this is the first description of a bacterial cryptochrome.
Asunto(s)
Cianobacterias/genética , ADN Bacteriano/efectos de la radiación , Desoxirribodipirimidina Fotoliasa/genética , Proteínas de Drosophila , Proteínas del Ojo , Flavoproteínas/genética , Células Fotorreceptoras de Invertebrados , Rayos Ultravioleta , Secuencia de Bases , Criptocromos , Cianobacterias/crecimiento & desarrollo , Cianobacterias/efectos de la radiación , Reparación del ADN , ADN Bacteriano/genética , Oscuridad , Genes Bacterianos , Luz , Datos de Secuencia Molecular , Filogenia , Receptores Acoplados a Proteínas GRESUMEN
Escherichia coli RuvB protein, together with RuvA, promotes branch migration of Holliday junctions during homologous recombination and recombination repair. The RuvB molecular motor is an intrinsic ATP-dependent DNA helicase with a hexameric ring structure and its architecture has been suggested to be related to those of the members of the AAA+ protein class. In this study, we isolated a large number of plasmids carrying ruvB mutant genes and identified amino acid residues important for the RuvB functions by examining the in vivo DNA repair activities of the mutant proteins. Based on these mutational studies and amino acid conservation among various RuvBs, we identified 10 RuvB motifs that agreed well with the features of the AAA+ protein class and that distinguished the primary structure of RuvB from that of typical DNA/RNA helicases with seven conserved helicase motifs.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Helicasas/genética , Reparación del ADN , Escherichia coli/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Secuencia Conservada , ADN Helicasas/química , ADN Helicasas/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/efectos de la radiación , Escherichia coli/enzimología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Rayos UltravioletaRESUMEN
The 1H-NMR spectra of a series of pyridylamino (PA-) derivatives of oligosaccharides were obtained and compared with those of the corresponding asparagine-linked sugar chains in order to elucidate the effect of the PA-group on the chemical shifts of structural-reporter signals. The effects were found to be localized within the two residues from the end group. Thus, the data for asparagine-linked chains in the literature are applicable to PA-derivatives, so the combination of pyridylamination and NMR measurements greatly reduces the time required for structure analysis of sugar chains of glycoproteins, because the isolation and purification of the chains as PA-derivatives are easy and efficient.
Asunto(s)
Glicoproteínas , Oligosacáridos , Asparagina , Conformación de Carbohidratos , Secuencia de Carbohidratos , Hidrógeno , Espectroscopía de Resonancia MagnéticaRESUMEN
An asparagine-linked sugar chain of a protease inhibitor from barbados pride (Caesalpinia pulcherrima Sw.) was liberated by hydrazinolysis. After N-acetylation, the reducing end residue of this carbohydrate unit was coupled with 2-aminopyridine and the pyridylamino (PA-) derivative was purified by gel-filtration and reversed-phase HPLC. The structure of the resulting PA-sugar chain was determined mainly by stepwise exoglycosidase digestions and 500 MHz 1H-NMR spectroscopy and proved to be as follows: (formula; see text).