Genetic and biochemical characterization of Salmonella enterica serovar typhi deoxyribokinase.
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.
Tourneux
L
L
Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, 75724 Paris Cedex 15, France.
Bucurenci
N
N
Saveanu
C
C
Kaminski
P A
PA
Bouzon
M
M
Pistotnik
E
E
Namane
A
A
Marlière
P
P
Bârzu
O
O
Li De La Sierra
I
I
Neuhard
J
J
Gilles
A M
AM
D000595:Amino Acid Sequence; D002874:Chromosome Mapping; D003855:Deoxyribose / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D005819:Genetic Markers; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D012485:Salmonella typhi / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship
D005819:Genetic Markers; D003855:Deoxyribose; D017853:Phosphotransferases (Alcohol Group Acceptor); C022534:ribokinase