Purification and characterization of a fusion protein of plant acetohydroxy acid synthase and acetohydroxy acid isomeroreductase.
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.
Dumas
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Unité Mixte CNRS/Rhône-Poulenc Agrochimie (UMR 41), Lyon, France.
Biou
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Douce
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D015072:2-Acetolactate Mutase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000095:Acetolactate Synthase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D000095:Acetolactate Synthase; D015072:2-Acetolactate Mutase