RESUMO
Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (Km = 23 microM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35 degrees C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the alpha/beta barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively.
Assuntos
Oxirredutases/genética , Rhizobium/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Dados de Sequência Molecular , Oxirredutases/isolamento & purificação , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
By library screening and PCR we have obtained cDNA clones which encode the gamma subunit of the CCT chaperonin complex from Xenopus laevis. The gene (XlCctg), which encodes the CCT gamma subunit contains an open reading frame which codes for 547 amino acid residues (60 kDa) and the predicted amino acid sequence shares a high degree of sequence identity with other CCT gamma homologues. The XlCctg mRNA measures 2.1 kb and is expressed ubiquitously in all of the X. laevis tissues examined. The mRNA levels of XlCctg are significantly higher in the ovary compared with other tissues.
Assuntos
Chaperoninas/genética , Genes/genética , Xenopus laevis/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Chaperonina com TCP-1 , DNA Complementar/genética , Feminino , Expressão Gênica , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/análise , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
We report the sequence analysis of a Drosophila melanogaster (Dm) P1 genomic clone (DS05563) which contains the gamma-chaperonin-encoding gene, Cctg. The (Hs) Cctg orthologue was found to share strong sequence identity with a 1603-bp region of DS05563, suggesting that Dm Cctg is located within this region. Detailed analysis has shown that Dm Cctg comprises four exons and is interrupted by three introns of 55, 85 and 66 bp. Dm Cctg encodes a predicted peptide of 545 amino acids (aa) (approx. 60 kDa). The predicted Dm CCT gamma aa sequence shares a high degree of sequence identity with gamma-orthologues from human (70%), mouse (70%), protozoa (60%) and yeast (60%), and also contains domains found in other chaperonins including bacterial GroEL, mitochondrial Hsp60 and plant Rubisco large subunit-binding protein. These data support the conclusion that the DS05563 clone contains the Dm Cctg gene.
Assuntos
Chaperoninas/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Sequência de Bases , Chaperonina 60/genética , Chaperonina com TCP-1 , Chaperoninas/química , Sequência Conservada , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
We describe the cloning, DNA sequence analysis and mRNA expression analysis of human Cctg (HsCctg), a gene that encodes the gamma-subunit of the eukaryotic cytosolic 'chaperonin-containing TCP-1' (CCT). Partial clones representing the 3' region of HsCctg cDNA were isolated from a human kidney cDNA library, and the missing 5' region was amplified directly from human kidney cDNA. The Cctg mRNA transcript is expressed in numerous human and mouse tissues and, like Tcp-1/Ccta, Cctg mRNA is expressed at higher levels in mouse testis when compared with kidney and brain. Southern-blot analysis has also revealed the Cctg gene to be highly conserved in mouse, rat, sheep and frog. The 1901 bp HsCctg cDNA has a coding region of 1635 bp and encodes a predicted 60 kDa protein (544 amino acids). The predicted HsCCT gamma amino acid sequence shares a high degree of sequence similarity with gamma-subunits from the mouse Mus musculus (98% similarity), the yeast Saccharomyces cerevisiae (75% similarity) and the protozoan Tetrahymena pyriformis (76% similarity) as well as with other members of the TF55/TCP-1 family, such as human TCP-1/CCT alpha (55% similarity) and TCP-20/CCT zeta (54% similarity). HsCCT gamma also shares conserved domains previously identified in the TF55/TCP-1 family of chaperonins and more distantly related chaperonins such as GroEL and Hsp60.
