RESUMO
Two cDNA clones (Frk1 and Frk2) encoding fructokinase (EC 2.7.1.4) were isolated from tomato (Lycopersicon esculentum). The Frk2 cDNA encoded a deduced protein of 328 amino acids that was more than 90% identical with a previously characterized potato (Solanum tuberosum) fructokinase. In contrast, the Frk1 cDNA encoded a deduced protein of 347 amino acids that shared only 55% amino acid identity with Frk2. Both deduced proteins possessed and ATP-binding motif and putative substrate recognition site sequences identified in bacterial fructokinases. The Frk1 cDNA was expressed in a mutant yeast (Saccharomyces cerevisiae) line, which lacks the ability to phosphorylate glucose and fructose and is unable to grow on glucose or fructose. Mutant cells expressing Frk1 were complemented to grow on fructose but not glucose, indicating that Frk1 phosphorylates fructose but not glucose, and this activity was verified in extracts of transformed yeast. The mRNA corresponding to Frk2 accumulated to high levels in young, developing tomato fruit, whereas the Frk1 mRNA accumulated to higher levels late in fruit development. The results indicate that fructokinase in tomato is encoded by two divergent genes, which exhibit a differential pattern of expression during fruit development.
Assuntos
Frutoquinases/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Solanum lycopersicum/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar , Frutoquinases/química , Frutoquinases/genética , Solanum lycopersicum/genética , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência de Aminoácidos , Solanum tuberosum/enzimologiaAssuntos
Proteínas de Transporte , Cromossomos Humanos Par 2 , Frutoquinases/genética , Proteínas/genética , Proteínas Adaptadoras de Transdução de Sinal , Processamento Alternativo , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Clonagem Molecular , Inibidores Enzimáticos/química , Escherichia coli , Frutoquinases/biossíntese , Frutoquinases/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Proteínas/química , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
The metabolic effects of the administration of fructose to a yeast expressing the cDNA for rat liver ketohexokinase have been investigated by 31P-nuclear magnetic resonance spectroscopy. Cessation of growth suffered by the yeast on exposure to 5 and 25 mM-fructose was accompanied by a large accumulation of fructose 1-phosphate at the expense of cytoplasmic orthophosphate and nucleoside triphosphate. Shifts in resonances were consistent with a drop in cytoplasmic pH. Arresting growth with 1 mM-fructose, however, did not result in these changes, although a large accumulation of fructose 1-phosphate occurred which may have been supported by the mobilization of polyphosphates.
Assuntos
Frutoquinases/biossíntese , Frutose/farmacologia , Saccharomyces cerevisiae/metabolismo , Animais , Frutoquinases/genética , Intolerância à Frutose , Espectroscopia de Ressonância Magnética , Isótopos de Fósforo , Ratos , Proteínas Recombinantes/biossíntese , Saccharomyces cerevisiae/efeitos dos fármacos , Transformação GenéticaRESUMO
The frk gene encoding the enzyme fructokinase (fructose 6-phosphotransferase [EC 2.7.1.4]) from Zymomonas mobilis has been isolated on a partial TaqI digest fragment of the genome and sequenced. An open reading frame of 906 bp corresponding to 302 amino acids was identified on a 3-kbp TaqI fragment. The deduced amino acid sequence corresponds to the first 20 amino acids (including an N-terminal methionine) determined by amino acid sequencing of the purified protein. The 118 bp preceding the methionine codon on this fragment does not appear to contain a promoter sequence. There was weak expression of the active enzyme in the recombinant Escherichia coli clone under control of the lac promoter on the pUC plasmid. Comparison of the amino acid sequence with that of the glucokinase enzyme (EC 2.7.1.2) from Z. mobilis reveals relatively little homology, despite the fact that fructokinase also binds glucose and has kinetic and structural properties similar to those of glucokinase. Also, there is little homology with hexose kinases that have been sequenced from other organisms. Northern (RNA) blot analysis showed that the frk transcript is 1.2 kb long. Fructokinase activity is elevated up to twofold when Z. mobilis was grown on fructose instead of glucose, and there was a parallel increase in frk mRNA levels. Differential mRNA stability was not a factor, since the half-lives of the frk transcript were 6.2 min for glucose-grown cells and 6.6 min for fructose-grown cells.
Assuntos
Frutoquinases/genética , Bacilos Gram-Negativos Anaeróbios Facultativos/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Frutoquinases/biossíntese , Frutose/metabolismo , Glucoquinase/genética , Glucose/metabolismo , Bacilos Gram-Negativos Anaeróbios Facultativos/enzimologia , Hexoses/metabolismo , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fosfotransferases/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
Growth on lactose by strains of Streptococcus mutans resulted in the induction of the lactose-phosphoenolpyruvate-phosphotransferase system, phospho-beta-galactosidase, and the enzymes of the tagatose 6-phosphate pathway.
