Purification and characterization of a fusion protein of plant acetohydroxy acid synthase and acetohydroxy acid isomeroreductase

FEBS Letters
R DumasR Douce

Abstract

The nucleotide sequence coding for the Arabidopsis thaliana acetohydroxy acid synthase was genetically fused in frame with the nucleotide sequence coding for the Spinacia oleracea acetohydroxy acid isomeroreductase and expressed in Escherichia coli. This construction allowed the production of large amounts of soluble fusion protein. The pure chimeric enzyme exhibits high acetohydroxy acid synthase and acetohydroxy acid isomeroreductase specific activities. Fusion and native enzymes exhibit similar Km values for their substrates and for most cofactors. Furthermore, whereas native plant acetohydroxy acid synthase is highly unstable, the stability of this enzyme in the fusion has been increased. Thus, the chimeric enzyme appears to be a useful tool for the determination of kinetic and structural properties of plant acetohydroxy acid synthase.

References

Jan 1, 1992·Analytical Biochemistry·W P Deng, J A Nickoloff
May 1, 1974·Analytical Biochemistry·R K Scopes
Jan 1, 1980·Methods in Enzymology·K E Neet, G R Ainslie
Jul 1, 1995·The Plant Cell·B Singh, D L Shaner
Jun 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·J K SmithB J Mazur

Related Concepts

Acetohydroxyacid Synthetase I
SDS-PAGE
Alkalescens-Dispar Group
Hybrid Proteins, Recombinant
2-Acetolactate Mutase
Gene Expression
Nested Polymerase Chain Reaction
Mutagenesis, Site-Directed
Arabidopsis thaliana <plant>

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