Crystallographic and analytical ultracentrifugation studies of the E. coli acetohydroxy acid synthase I enzyme

Abstract

This thesis ellucidates the mechanism of activation and regulation of AHAS I enzyme from Escherichia coli that was characterised using crystallographic and sedimentation velocity analytical ultracentrifugation studies. AHAS I is a large multi-subunit enzyme that was discovered in the 1950’s, by Umbarger, as the enzyme that catalyses the first committed step in the biosynthesis of branched chain amino acids, isoleucine, leucine and valine in bacteria, plants and certain fungi. The enzyme itself is composed of a catalytic (large) subunit (CSU / LSU) and a regulatory (small) subunit (RSU / SSU) that are coded for by the genes IlvB and IlvN, respectively. The enzyme also requires TPP, FAD and Mg2+ as co-factors. The enzyme catalyses the thiamine dependent decarboxylation of pyruvate followed by an “acyloin like" condensation with either another molecule of pyruvate or with 2-ketobutyrate. The product of the former condensation is acetolactate, while that of the later is 2-keto-2-hydroxybutyrate, which are precursors for the synthesis of valine (and leucine) and isoleucine, respectively. Feedback regulation occurs via binding of valine or isoleucine to the RSU, wherein the amino acids are negative and positive effectors of the enzyme, respectively. The LSU’s are well conserved across species. The RSU’s on the other hand differ significantly in sequence and size across species and in the selectivity towards inhibition by one of the branched chain amino acids. The catalytic and enzymatic properties of this enzyme have been studied in great detail. Despite this rich history, structures of the AHAS holoenzyme from any species have been elusive and as a consequence the structural basis for the activation and regulation of this enzyme remain unclear at best. It has been surmised that the intrinsic instability of the holoenzyme complex, as a result of weak interactions between the AHAS subunits, results in heterogeneity of the holoenzyme preparation and that this is the major factor affecting its crystallisation. On the other hand structural characterization of the holoenzyme using solution NMR methods is a daunting task due to the inherent size of the complex. However the RSU has been successfully cloned, purified and characterized using NMR. Important information regarding the behaviour of IlvN in the effector bound and free state has been elucidated. The structural differences as observed for IlvN in the presence of valine as opposed to isoleucine have also been partially characterised. Even with the availability of these data, understanding the difference in affinity exhibited by IlvN for the two effector molecules remains undescribed due to the lack of structures elucidating effector binding and its interactions with residues in the binding pocket. This thesis describes the crystallographic and analytical ultracentrifugation studies of the subunits of the E.coli AHAS I enzyme and of the AHAS I holoenzyme. The crystallographic studies were carried out to understand at atomic resolution the conformational factors that determine selectivity for the effector molecules. The hydrodynamic studies were carried out to understand the solution conformational properties and the oligomeric status of the subunits in solution and the stoichiometry of the subunits in the catalytically active holoenzyme.