The fructokinase from Rhizobium leguminosarum biovar trifolii belongs to group I fructokinase enzymes and is encoded separately from other carbohydrate metabolism enzymes

The Rhizobium leguminosarum by. trifolii BAL fructokinase (frk) gene was isolated on a 2.4 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-N-16-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 36.5 kDa, pl of 4.65, ph activity range of 6.0-9.0 (maximum activity at ph 8.0) and a Mg2+ requirement. It had a K-m of 0.31 mM and a V-max of 31 mu mol fructose 6-phosphate (mg protein)(-1) min(-1) with fructose as substrate. The R. leguminosarum by. trifolii BAL fructokinase was biochemically and molecularly similar to other bacterial fructokinases.