Genetic control of manno(fructo)kinase activity in Escherichia coli.
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 degrees 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.
Sproul
A A
AA
Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA.
Lambourne
L T
LT
Jean-Jacques D
J
J
Kornberg
H L
HL
D002874:Chromosome Mapping; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005631:Fructokinases / Q000378:metabolism*; D008040:Genetic Linkage; D016680:Genome, Bacterial; D009154:Mutation
D005631:Fructokinases; C027295:mannofructokinase