Affinity purification of 5-methylthioribose kinase and 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Klebsiella pneumoniae [corrected].
Two enzymes in the methionine salvage pathway, 5-methylthioribose kinase (MTR kinase) and 5'-methylthioadenosine/ S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) were purified from Klebsiella pneumoniae. Chromatography using a novel 5'-(p-aminophenyl)thioadenosine/5-(p-aminophenyl)thioribose affinity matrix allowed the binding and selective elution of each of the enzymes in pure form. The molecular mass, substrate kinetics and N-terminal amino acid sequences were characterized for each of the enzymes. Purified MTR kinase exhibits an apparent molecular mass of 46-50 kDa by SDS/PAGE and S200HR chromatography, and has a Km for MTR of 12.2 microM. Homogeneous MTA/SAH nucleosidase displays a molecular mass of 26.5 kDa by SDS/PAGE, and a Km for MTA of 8.7 microM. Comparisons of the N-terminal sequences obtained for each of the enzymes with protein-sequence databases failed to reveal any significant sequence similarities to known proteins. However, the amino acid sequence obtained for the nucleosidase did share a high degree of sequence similarity with the putative translation product of an open reading frame in Escherichia coli, thus providing a tentative identification of this gene as encoding an MTA/SAH nucleosidase.
Cornell
K A
KA
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201, USA.
Winter
R W
RW
Tower
P A
PA
Riscoe
M K
MK
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives; D017421:Sequence Analysis
D012685:Sepharose; D017853:Phosphotransferases (Alcohol Group Acceptor); C021787:5-methylthioribose kinase; D009699:N-Glycosyl Hydrolases; C019606:adenosylhomocysteine nucleosidase