Purification of Escherichia coli acetohydroxyacid synthase isoenzyme II and reconstitution of active enzyme from its individual pure subunits.
The first step in the biosynthesis of branched-chain amino acids is catalysed by acetohydroxyacid synthase (EC 4.1.3.18). The reaction involves the decarboxylation of pyruvate followed by condensation with either a second molecule of pyruvate or with 2-oxobutyrate. The enzyme requires as cofactors thiamine diphosphate, a divalent metal ion and, usually, FAD. In most bacteria the enzyme is a heterotetramer of two large and two small subunits. Escherichia coli contains three active isoenzymes and the present study concerns isoenzyme II, whose large and small subunits are encoded by the ilvG and ilvM genes respectively. Cloning these genes into a plasmid vector and overexpression in E. coli allowed a two-step purification procedure for the native enzyme to be developed. The level of expression is considerably higher from a vector that introduces a 50 residue N-terminal fusion containing an oligohistidine sequence on the large subunit. Purification to homogeneity was achieved in a single step by immobilized-metal-affinity chromatography. The kinetic properties of the native and fusion enzyme are indistinguishable with respect to the substrate pyruvate and the inhibitor chlorsulfuron. The individual subunits were expressed as oligohistidine-tagged fusion proteins and each was purified in a single step. Neither subunit alone has significant enzymic activity but, on mixing, the enzyme is reconstituted. The kinetic properties of the reconstituted enzyme are very similar to those of the fusion enzyme. It is proposed that the reconstitution pathway involves successive, and highly co-operative, binding of two small subunit monomers to a large subunit dimer. None of the cofactors is needed for subunit association although they are necessary for the restoration of enzymic activity.
Hill
C M
CM
Centre for Protein Structure, Function and Engineering, Department of Biochemistry, University of Queensland, Brisbane 4072, Australia.
Pang
S S
SS
Duggleby
R G
RG
D000095:Acetolactate Synthase / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000201:enzymology*; D005798:Genes, Bacterial; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D000095:Acetolactate Synthase