Expression, purification and characterization of Arabidopsis thaliana acetohydroxyacid synthase.
Acetohydroxyacid synthase (EC 4.1.3.18) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine. The AHAS gene from Arabidopsis thaliana with part of the chloroplast transit sequence removed was cloned into the bacterial expression vector pT7-7 and expressed in the Escherichia coli strain BL21(DE3). The expressed enzyme was purified by an extensive procedure involving (NH4)2SO4 fractionation followed by hydrophobic and anion-exchange chromatography. The purified enzyme appears as a single band on SDS/PAGE with a molecular mass of about 61 kDa. On gel filtration the enzyme is a dimer, migrating as a single peak with molecular masses of 109 and 113 kDa in the absence and presence of FAD respectively. Ion spray MS analysis yielded a mass of 63864 Da. The enzyme has optimum activity in the pH range 6.5-8.5 and exhibits absolute dependence on the three cofactors FAD, Mg2+ and thiamine diphosphate for activity. It displays negatively co-operative kinetics with respect to pyruvate concentration. A model was derived to explain the non-hyperbolic substrate-saturation curve, involving interaction between the active sites of the dimer. The Km for the first active site was found to be 8.01 +/- 0.66 mM; the Km for the second active site could not be accurately determined but was estimated to be approx. 100 mM. The enzyme is insensitive to valine, leucine and isoleucine but is strongly inhibited by the sulphonylurea herbicide, chlorsulphuron, and the imidazolinone herbicide, imazapyr. Inhibition by both herbicides exhibits slow-binding kinetics, whereas chlorsulphuron also shows tight-binding inhibition.
Chang
A K
AK
Centre for Protein Structure, Function and Engineering, Department of Biochemistry, University of Queensland, Brisbane, QLD 4072, Australia.
Duggleby
R G
RG
D000095:Acetolactate Synthase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D015870:Gene Expression; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009525:Niacin / Q000031:analogs & derivatives / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry; D019289:Pyruvic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D013449:Sulfonamides; D013835:Thiamine Pyrophosphate / Q000494:pharmacology; D014227:Triazines / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007093:Imidazoles; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013449:Sulfonamides; D014227:Triazines; D005182:Flavin-Adenine Dinucleotide; D009525:Niacin; C076656:imazapyr; D019289:Pyruvic Acid; D000095:Acetolactate Synthase; C037137:chlorsulfuron; D013835:Thiamine Pyrophosphate