alexa Affinity purification of 5-methylthioribose kinase and 5-methylthioadenosine S-adenosylhomocysteine nucleosidase from Klebsiella pneumoniae [corrected].
Biochemistry

Biochemistry

Biochemistry & Analytical Biochemistry

Author(s): Cornell KA, Winter RW, Tower PA, Riscoe MK

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Abstract 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.
This article was published in Biochem J and referenced in Biochemistry & Analytical Biochemistry

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