RESUMEN
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic pathway that catalyzes the conversion of D-glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate. Here, the full-length GAPDH type 1 from Escherichia coli (EcGAPDH1) was cloned and overexpressed, and the protein was purified. Biochemical analyses found that the optimum reaction temperature and pH of EcGAPDH1 were 55°C and 10.0, respectively. The protein has a certain amount of thermostability. Crystals of EcGAPDH1 were obtained using the sitting-drop vapor-diffusion technique and X-ray diffraction data were collected to 1.88â Å resolution. Characterization of the crystals showed that they belonged to space group P41212, with unit-cell parameters a = b = 89.651, c = 341.007â Å, α = ß = γ = 90°. The structure of EcGAPDH1 contains four subunits, each of which includes an N-terminal NAD+-binding domain and a C-terminal catalytic domain. Analysis of the NAD+-bound form showed some differences between the structures of EcGAPDH1 and human GAPDH. As EcGAPDH1 shares 100% identity with GAPDH from Shigella sonnei, its structure may help in finding a drug for the treatment of shigellosis.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Gliceraldehído 3-Fosfato/química , Gliceraldehído-3-Fosfato Deshidrogenasas/química , NAD/química , Subunidades de Proteína/química , Secuencia de Aminoácidos , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Gliceraldehído 3-Fosfato/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Modelos Moleculares , NAD/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Shigella sonnei/enzimología , Shigella sonnei/genéticaRESUMEN
ARM genes, whose polypeptide consist of Armadillo/beta-catenin-like repeats (ARM) domain(s), exist ubiquitously from fly to vertebrates. These genes have multiple functions in signal transduction, development, cell adhesion and mobility, tumor initiation and metastasis. In this study, we have isolated a novel splicing variant of ARMC3 from human fetal brain, which is 2439 bp, encoding a 688-amino acid polypeptide that contains three typical ARM domains. The cDNA called ARMC3_v2 and the original called ARMC3_v1 (GeneBank: BC039312) are both located on the human chromosome 10p12.23. RT-PCR analysis in our work showed that ARMC3_v2 was detected in human skeletal muscle, liver, spleen and thymus; in contrast, ARMC3_v1 in skeletal muscle, lung, prostate and testis.
Asunto(s)
Proteínas del Dominio Armadillo/genética , Empalme del ARN , Secuencia de Aminoácidos , Proteínas del Dominio Armadillo/biosíntesis , Secuencia de Bases , Encéfalo/metabolismo , Cromosomas Humanos Par 10/genética , Clonación Molecular , Feto , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos , Estructura Terciaria de ProteínaRESUMEN
In order to investigate the effects of amino acid replacement on the characters of thermostable catechol 2, 3-dioxygenase, two mutants (Pro229Ser and Glu243Gly) of this enzyme were obtained by using the method of PCR random mutagenesis. The wild type thermostable catechol 2, 3-dioxygenase and these two mutants (Pro229Ser, Glu243Gly) were over expressed in E. coli TG1 and purified. The enzymatic characters and thermostability of the wild type enzyme and the two mutants (Pro229Ser, Glu243Gly) were analyzed. The results revealed that the optimum enzymatic temperature of the two mutants were the same as that of the wild type enzyme (60 degrees C) and the Kcat/Km value of Pro229Ser and Glu243Gly (4.89 +/- 0.01 x 10(6) mol-1 s-1 and 5.88 +/- 0.01 x 10(6) mol-1 s-1, respectively) were reduced compared with the wild type enzyme (6.97 +/- 0.01 x 10(6) mol-1 s-1). However, the thermostability of Pro229Ser extremely decreased 10.2 degrees C and the thermostability of Glu243Gly slightly increased 1.5 degrees C. It was proposed that Pro229 played an important role on the thermostability of thermostable catechol 2, 3-dioxygenase.
Asunto(s)
Dioxigenasas , Oxigenasas/metabolismo , Secuencia de Aminoácidos , Catálisis , Catecol 2,3-Dioxigenasa , Estabilidad de Enzimas , Calor , Cinética , Datos de Secuencia Molecular , Oxigenasas/química , Mutación Puntual , Relación Estructura-ActividadRESUMEN
Through PCR-mediated mutagenesis, three mutants E68S, S70A and E68SS70A around active site S(69) were obtained. Their enzymatic characteristics was determined. It was found that the specific activity of E68S ascended 8 times while its optional reactive temperature climbed 20 degrees C and its Tm descended 3 degrees C; the specific activity of S70A ascended 1 time while its optional reactive temperature climbed 5 degrees C and its Tm descended 2 degrees C; the specific activity of E68SS70A descended 50% while its optional reactive temperature climbed 5 degrees C and its Tm descended 19 degrees C. These result implied that the amino acids, beside the active site, were contributed not only to enzymatic activity but also to its thermostability and thermophilicity. The work provided the direction for mutation to improve enzymatic specific activity and studying the mechanism of thermostability and thermophilicity.
Asunto(s)
Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Sitios de Unión , Estabilidad de Enzimas , Calor , Mutagénesis Sitio-Dirigida , Relación Estructura-ActividadRESUMEN
Using the method of double primer oligonucleotide-mediated mutagenesis, the high expression plasmid of TaqND236, a derivative of Taq DNA polymerase, was constructed. To determine the frameshift mutation frequency of the in vitro DNA synthesis, we constructed a Gapped-DNA system using the pFDPM118 (a mutant of pUC118 with a -1 frameshift mutation on the lacZ gene) as template. By calculating the ratio of blue and white colonies on the X-gal plate after transforming E. coli TG1, the frameshift mutation frequency of Taq and TaqND236 was measured. It was found that the replication fidelity of the deleted Taq-TaqND236 increased more than 10 folds.
Asunto(s)
Polimerasa Taq/metabolismo , Replicación del ADN , Escherichia coli/genética , Mutación del Sistema de Lectura , Polimerasa Taq/genéticaRESUMEN
To reveal the mechanism of protein thermostability, we used in vivo random mutagenesis to generate variants of pTAP503F which contained thermostable alkaline phosphatase (FD-TAP). After screening about 5,000 clones, we obtained 4 temperature-sensitive mutants. The study of enzymatic properties of one mutant (TAPM3) showed that the thermostability of the mutant enzyme descended a lot, compared to the wild type, while the thermoactivity remained stable. DNA sequencing showed that the G-A transition in position 1,239 resulted in the substitution from glysine to serine in position 427. This mutation conspicuously affected thermostability, Michaelis constant and energy of activation. This suggests that only one substitution of amino acid will make great changes in thermostability and other properties, meanwhile, side-chain size, charge of residues and so on, which loosen the structure of protein, will result in the descent of thermostability.
Asunto(s)
Fosfatasa Alcalina/genética , Fosfatasa Alcalina/química , Aminoácidos , Calor , MutaciónRESUMEN
Thermostable alkaline phosphatase from Thermus sp. 3041 has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P2(1)22(1), with unit-cell parameters a = 57.7, b = 69.9, c = 111.5 A. Diffraction data were collected to 2.54 A with a completeness of 91.1% (87.8% for the last shell), an R(merge) value of 0.105 (0.312) and an I/sigma(I) value of 9.5 (3.6).