Genetic control of manno(fructo)kinase activity in Escherichia coli
AUTOR(ES)
Sproul, Andrew A.
FONTE
The National Academy of Sciences
RESUMO
Mutants of Escherichia coli unable to use fructose by means of the phosphoenolpyruvate/glycose phosphotransferase system mutate further to permit growth on that ketose by derepression of a manno(fructo)kinase (Mak+ phenotype) present in only trace amounts in the parent organisms (Mak-o phenotype). The mak gene was located at min 8.8 on the E. coli linkage map as an ORF designated yajF, of hitherto unknown function; it specifies a deduced polypeptide of 344 aa. The derepression of Mak activity was associated with a single base change at position 71 (codon 24) of the gene, where GCC (alanine) in Mak-o has been changed to GAC (aspartate) in Mak+. By cloning selected portions of the total 1,032-bp mak gene into a plasmid that also carried a temperature-sensitive promoter, we showed that the mutation resided in a 117-bp region that does not specify sequences necessary for Mak activity but was located 46 bp upstream of a 915-bp portion that does. Mak+ and Mak-o strains differ greatly in the heat stability of the enzyme: at 61°C, mak-o cloned into a mak-o recipient loses 50% of its activity in approximately 6 min, whereas it takes over 30 min to achieve a similar reduction in the activity of mak+ cloned into a mak-o strain. However, the Mak activity of the cloned fragment specifying the enzyme without the regulatory region lost activity with a half-life of 29 min irrespective of whether it was derived from a mak+ or a mak-o donor, which indicates that the A24D mutation contributes to the high enzyme activity of Mak+ mutants by serving to protect Mak from denaturation.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=65016Documentos Relacionados
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