Genetic and biochemical characterization of Salmonella enterica serovar Typhi deoxyribokinase

We identified in the genome of Salmonella enterica serovar Typhi the gene e ncoding deoxyribokinase, deoK, Two other genes, vicinal to deoK, were deter mined to encode the putative deoxyribose transporter (deoP) and a repressor protein (deoQ), This locus, located between the uhpA and ilvN genes, is ab sent in Escherichia coli, The deoK gene inserted on a plasmid provides a se lectable marker in E. coli for growth on deoxyribose-containing medium. Deo xyribokinase is a 306-amino-acid protein which exhibits about 35% identity with ribokinase from serovar Typhi, S. enterica serovar Typhimurium, or E, coli, The catalytic properties of the recombinant deoxyribokinase overprodu ced in E, coli correspond to those previously described for the enzyme isol ated from serovar Typhimurium. From a sequence comparison between serovar T yphi deoxyribokinase and E. coli ribokinase, whose crystal structure was re cently solved, we deduced that a key residue differentiating ribose and deo xyribose is Met10, which in ribokinase is replaced by Asnl4, Replacement by site-directed mutagenesis of Met10 with Asn decreased the V-max of deoxyri bokinase by a factor of 2.5 and increased the K-m for deoxyribose by a fact or of 70, compared to the parent enzyme.