Engineering and kinetic characterisation of two glucosyltransferases from Arabidopsis thaliana.
This study describes the characterisation of a chimeric mutant derived from two arabidopsis glucosyltransferases, 71C1 and 71C3. A chimera, N1C3, was constructed to contain the N-terminal domain of 71C1 and the C-terminal domain of 71C3. The chimera and the wild-type GTs displayed a similar Km towards the acceptor scopoletin. However, N1C3 had a Km near identical to 71C3 towards UDP-glucose, but was three-fold lower than that of 71C1. The results suggest that the acceptor and sugar donor are recognised independently by the N- and C-terminal domain of the GTs respectively, and provide a foundation for the future design of glucosyltransferase biocatalysts through assembling domains with different affinity towards the acceptor and donor.
Weis
Markus
M
CNAP, Department of Biology (Area 8), University of York, York, YO10 5YW, UK.
Lim
Eng-Kiat
EK
Bruce
Neil C
NC
Bowles
Dianna J
DJ
D017360:Arabidopsis / Q000201:enzymology*; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D005964:Glucosyltransferases