THE FRUCTOKINASE FROM RHIZOBIUM-LEGUMINOSARUM BIOVAR TRIFOLII BELONGSTO GROUP-I FRUCTOKINASE ENZYMES AND IS ENCODED SEPARATELY FROM OTHER CARBOHYDRATE-METABOLISM ENZYMES

The Rhizobium leguminosarum by. trifolii BAL fructokinase (frk) gene w as isolated on a 2.4 kb BamHI fragment from the cosmid pLA72 by comple mentation 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 sequenc es. The fructokinase gene was not contained in an operon and is encode d separately from other enzymes of carbohydrate metabolism. Its produc t is therefore assigned to the group I fructokinases. A putative promo ter (TTGACA-N-16-GTTGAT), ribosome-binding site and termination sequen ce 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 gen e sequence data revealed the fructokinase as a hydrophilic protein. Th e fructokinase protein was purified to electrophoretic homogeneity by a three-step method using chromatofocusing, affinity chromatography an d gel filtration. Its purity was confirmed by SDS-PAGE and it was visu alized as a single band by silver staining. The N-terminal amino acid sequence of the purified fructokinase confirmed the proposed open read ing frame of the frk gene. The purified fructokinase had a molecular m ass of 36.5 kDa, pl of 4.65, ph activity range of 6.0-9.0 (maximum act ivity 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 fructoki nase was biochemically and molecularly similar to other bacterial fruc tokinases.