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 ca talysed by acetohydroxyacid synthase (EC 4.1.3.18). The reaction invol ves the decarboxylation of pyruvate followed by condensation with eith er a second molecule of pyruvate or with 2-oxobutyrate. The enzyme req uires as cofactors thiamine diphosphate, a divalent metal ion and, usu ally, FAD. In most bacteria the enzyme is a heterotetramer of two larg e and two small subunits. Escherichia coli contains three active isoen zymes and the present study concerns isoenzyme II, whose large and sma ll subunits are encoded by the ilvG and ilvM genes respectively. Cloni ng these genes into a plasmid vector and overexpression in E. coli all owed a two-step purification procedure for the native enzyme to be dev eloped. The level of expression is considerably higher from a vector t hat introduces a 50 residue N-terminal fusion containing an oligohisti dine sequence on the large subunit. Purification to homogeneity was ac hieved in a single step by immobilized-metal-affinity chromatography. The kinetic properties of the native and fusion enzyme are indistingui shable with respect to the substrate pyruvate and the inhibitor chlors ulfuron. The individual subunits were expressed as oligohistidine-tagg ed fusion proteins and each was purified in a single step. Neither sub unit 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 th e 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 n ecessary for the restoration of enzymic activity.