Ketol-acid reductoisomerase (EC 188.8.131.52) is involved in the biosynthesis of the branched-chain amino acids, leucine, valine and isoleucine. It is a bifunctional enzyme that catalyzes two quite different reactions at a common active site; an isomerization consisting of an alkyl migration having a specific requirement for Mg2+, followed by an NADPH-dependent reduction of a 2-ketoacid, which can occur in the presence of Mg2+, Mn2+ or Co2+. The 2-ketoacid formed by the alkyl migration does not exist as an independent entity. KARl is the target of the experimental herbicides Hoe704 and IpOHA.
Amino acid sequence comparisons across species reveal that there are two types of KARl: a short form (Class I) found in fungi and most bacteria (except some ϒ-proteobacteria including Escherichia coli) and a long form (Class II) typical of plants. Crystal structures of each have been solved. The positions of the conserved active site residues are similar in the structures of both forms.
In this report, I have investigated the reaction mechanism of E. coli KARl using three complementary approaches; site-directed mutagenesis, crystal structure determination and in silico mutagenesis. Based on the position of the conserved active site residues E213, E389 and S414 in the structure, site directed mutagenesis studies were performed. The kinetic characterization of these mutants revealed that each of these residues has an important role in the catalysis. I have solved the crystal structure E. coli KARl to 2.6 A during this project. This is the first crystal structure of any Class II bacterial KARL. The enzyme shows an unusual arrangement of four monomers in the asymmetric unit comprising two halves of two tetramers. The biologically active tetrameric unit is generated by translating one of these dimers across a crystallographic symmetry axis. The overall structure is similar to the plant enzyme with an N-terminal α/β domain that resembles that found in other pyridine nucleotide dependent dehydrogenases, and an α-helical C-terminal domain that shows strong evidence of an internal duplication. All the active site residues in E. coli KARl superimpose very closely in three dimensional space on the plant and Pseudomonas aeruginosa KARl With the aim of correlating the site-directed mutagenesis studies and crystal structure of E. coli KARl, I have constructed the structures of the E213, E389 and S414 mutants in silico and minimization studies for wildtype and mutants are in the optimization stage.