Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.2.1.7 (BAL)
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Transposon Tn5 was used to generate a fructokinase mutation in Rhizobium leguminosarum biovar trifolii BAL. The section of the genome containing Tn5 was cloned into the EcoRI site of the vector pHC79 and isolated by direct selection on medium containing kanamycin and tetracycline. Total EcoRI digestion was used to obtain a single fragment containing Tn5 and flanking DNA sequences. The flanking DNA was used as a probe to isolate an intact fructokinase gene from a pLAFR1 cosmid clone bank of the parental strain. A cosmid showing homology to the probe was tri-parentally conjugated into the fructokinase-negative strain, complementing the mutation. The complemented mutant exhibited the wild-type phenotype, with an increase in fructokinase production presumably due to multiple copies of the gene.
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PMID:Transposon mutagenesis and complementation of the fructokinase gene in Rhizobium leguminosarum biovar trifolii. 256 Dec 89

The Rhizobium leguminosarum bv. trifolii BAL fructokinase (frk) gene was isolated on a 2 center dot 4 kb BamHI fragment from the cosmid pLA72 by complementation analysis of the Tn5-induced frk mutant BAL79, and confirmed by hybridization analysis. The nucleotide sequence of the frk gene was found to contain an open reading frame consisting of 978 bp encoding 326 amino acids, which was then compared to known fructokinase sequences. The fructokinase gene was not contained in an operon and is encoded separately from other enzymes of carbohydrate metabolism. Its product is therefore assigned to the group I fructokinases. A putative promoter (TTGACA-N16-GTTGAT), ribosome-binding site and termination sequence were identified. The Frk protein contained several motifs conserved in other known fructokinase sequences, including an ATP-binding and a substrate-binding motif. The hydropathy plot derived from the frk gene sequence data revealed the fructokinase as a hydrophilic protein. The fructokinase protein was purified to electrophoretic homogeneity by a three-step method using chromatofocusing, affinity chromatography and gel filtration. Its purity was confirmed by SDS-PAGE and it was visualized as a single band by silver staining. The N-terminal amino acid sequence of the purified fructokinase confirmed the proposed open reading frame of the frk gene. The purified fructokinase had a molecular mass of 36 center dot 5 kDa, pl of 4 center dot 65, pH activity range of 6 center dot 0-9 center dot 0 (maximum activity at pH 8 center dot 0) and a Mg2+ requirement. It had a Km of 0 center dot 31 mM and a Vmax of 31 mumol fructose 6-phosphate (mg protein)-1 min-1 with fructose as substrate. The R. leguminosarum bv. trifolii BAL fructokinase was biochemically and molecularly similar to other bacterial fructokinases.
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PMID:The fructokinase from Rhizobium leguminosarum biovar trifolii belongs to group I fructokinase enzymes and is encoded separately from other carbohydrate metabolism enzymes. 893 6