Cloning and expression of Escherichia coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase: identification of the pfs gene product.
The enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (EC 3.2.2.9) is responsible for cleavage of the glycosidic bond in both 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH). Based on amino acid sequence analysis of this enzyme from Klebsiella, we recently speculated that an open reading frame found in E. coli (designated pfs) encoded MTA/SAH nucleosidase. To explore this possibility, we amplified, cloned, and expressed the complete pfs gene from E. coli genomic DNA. The recombinant protein exhibited a molecular weight and Michaelis constants for MTA that are in agreement with those reported for native enzyme. From this biochemical evidence we confirm our original assignment of the pfs gene as encoding MTA/SAH nucleosidase.
Cornell
K A
KA
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201, USA.
Riscoe
M K
MK
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D009699:N-Glycosyl Hydrolases; C019606:adenosylhomocysteine nucleosidase