Genetic and Biochemical Characterization of Salmonella enterica Serovar Typhi Deoxyribokinase

Abstract

ABSTRACT We identified in the genome of Salmonella enterica serovar Typhi the gene encoding deoxyribokinase, deoK . Two other genes, vicinal to deoK , were determined to encode the putative deoxyribose transporter ( deoP ) and a repressor protein ( deoQ ). This locus, located between the uhpA and ilvN genes, is absent in Escherichia coli . The deoK gene inserted on a plasmid provides a selectable marker in E. coli for growth on deoxyribose-containing medium. Deoxyribokinase 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 overproduced in E. coli correspond to those previously described for the enzyme isolated from serovar Typhimurium. From a sequence comparison between serovar Typhi deoxyribokinase and E. coli ribokinase, whose crystal structure was recently solved, we deduced that a key residue differentiating ribose and deoxyribose is Met10, which in ribokinase is replaced by Asn14. Replacement by site-directed mutagenesis of Met10 with Asn decreased the V max of deoxyribokinase by a factor of 2.5 and increased the K m for deoxyribose by a factor of 70, compared to the parent enzyme.