Molecular analysis of two fructokinases involved in sucrose metabolism of enteric bacteria

Summary Sucrose-positive derivatives of Escherichia coli K-12, containing the plasmid pUR400, and of Kiebsieiia pneumoniae hydrolyse intracellular sucrose 6-phosphate by means of an invertase into D-glucose 6phosphate and free D-fructose. The latter is phosphorylated by an ATP-dependent fruclokinase (gene scrK of an ser reguion) to D-fructose 6-phosphate. The lack of ScrK does not cause any visible phenotype in wildtype strains of both organisms. Using genes and enzymes normally involved in D-arabinitoi metabolism from E. coli C and K. pneumoniae, derivatives of E. coii K-12 vi/ere constructed which allowed the identification of scr/C mutations on conventional indicator plates. Cloning and sequencing of scrK from sucrose ptasmid pUR400 and from the chromosome of K. pneumoniae revealed an open reading frame of 924 bp in both cases — the equivalent of a peptide containing 307 amino acid residues {M, 39 and 34 kDa, respectively, on sodium dodecyl sulphate gels). The sequences showed overall identity among each other (69% identical residues) and to a kinase from Vibrio alginoiyticus (57%) also involved in sucrose metabolism, iower overall identity (39%) to a Dribose-kinase from E. coli, and local similarity to prokaryotic, and eukaryotic phosphofructokinases at the putative ATP-binding sites.