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Cloning, sequencing, and expression of the Zymomonas mobilis fructokinase gene and structural comparison of the enzyme with other hexose kinases.

Centre for Protein and Enzyme Technology, La Trobe University, Bundoora, Victoria, Australia.

ABSTRACT

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.

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