Characterization and investigation of substrate specificity of the sugar aminotransferase WecE from E. coli K12.
WecE gene, encoding a sugar aminotransferase (SAT), has been cloned from E. coli K12 and expressed in E. coli BL21 (DE3). The enzyme was purified and characterized. WecE used TDP-4-keto-6-deoxy-D-glucose (TDP-D-Glc4O) and L-glutamate as a good amino acceptor and donor, respectively, leading to the production of TDP-4-amino-4,6-dideoxy-D-galactose (TDP-Fuc4N), which was identified by NMR studies. WecE also showed a similar activity for TDP-4-keto 6-deoxy-D-mannose (TDP-D-Man4O), but no activity for GDP-4-keto-6-deoxy-D-mannose (GDP-D-Man4O), suggesting that the nucleotide moiety would become a key determinant to the substrate specificity of amine acceptor for the activity of the SAT. Multiple alignments showed that SATs have four highly conserved motifs located around the active site and could be divided into three subgroups (VIalpha, VIbeta, and VIgamma) that might be closely related with their substrate specificities.
School of Chemical Engineering and Institute for Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Korea.
Bum-Yeol
BY
Hwang
Hwa-Jin
HJ
Lee
Yung-Hun
YH
Yang
Hwang-Soo
HS
Joo
Byung-Gee
BG
Kim
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D029968:Escherichia coli Proteins; D000637:Transaminases; C000608766:WecE protein, E coli