Purification, characterization and HPLC assay of Salmonella glucose-1-phosphate thymidylyl-transferase from the cloned rfbA gene.
We here report on the purification and characterization of glucose-1-phosphate thymidylyl-transferase, the first of four enzymes committed to biosynthesis of dTDP-L-rhamnose from Salmonella enterica strain LT2. The purification was greatly facilitated by the cloning of the rfbA gene encoding this enzyme. Pure enzyme was obtained by 109-fold enrichment in three chromatography steps. The glucose-1-phosphate thymidylytransferase catalyzes a reversible bimolecular group transfer reaction and kinetic measurements indicate that it acts by a 'ping-pong' mechanism. The Km values for dTTP and alpha -D-glucose 1-phosphate in the forward reaction are 0.020 mM and 0.11 mM, respectively. In the reverse reaction the Km values for dTDP-D-glucose and diphosphate are 0.083 mM and 0.15 mM, respectively. The enzyme also accepts UTP and UDP-D-glucose and alpha-D-glucosamine 1-phosphate is accepted equally as well as alpha-D-glucose 1-phosphate. The NH2-terminal sequence of glucose-1-phosphate thymidylyl-transferase agrees with the sequence predicted from the nucleotide sequence of the orf6.1 gene of the rfb gene cluster. The SDS/PAGE estimated subunit mass of 31 kDa agrees well with that calculated from the amino acid composition deduced from the nucleotide sequence of the orf6.1 gene (32453 Da).
Lindquist
L
L
Department of Clinical Bacteriology, Karolinska Institute, Huddinge Hospital, Sweden.
Kaiser
R
R
Reeves
P R
PR
Lindberg
A A
AA
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D005958:Glucosephosphates / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D012475:Salmonella / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism
D005958:Glucosephosphates; D013942:Thymine Nucleotides; C031590:glucose-1-phosphate; D009713:Nucleotidyltransferases; C020587:glucose-1-phosphate thymidylyltransferase; C024157:thymidine 5'-triphosphate