Chemistry Weekly Seminar - PhD Candidate Mohadeseh Majdi Yazdi

Title:

Understanding Allosteric Inhibition of Dihydrodipicolinate Synthase from Campylobacter jejuni

Abstract:

The diaminopimelate (DAP) pathway is responsible for de novo synthesis of L-lysine. meso-DAP is the immediate metabolic precursor and is a vital building block for bacterial cell wall synthesis. Some enzymes involved in the metabolism of amino acids, including dihydrodipicolinate synthase (DHDPS), are putative antibiotic targets. This is due to essential role of amino acids for bacterial growth, and absence of these anabolic pathways in animals and particularly humans. DHDPS is feedback-inhibited by L-lysine, which is the end-product representing key regulation point in the pathway.

This research investigates molecular details of the reaction catalyzed by DHDPS, and its mode of inhibition by lysine, bislysine and other novel inhibitors, with the aim of in-vitro reaction control and regulation. For this purpose, a number of DHDPS variants, with mutated key residues of the ligand-binding site, were studied to understand their effect on the first committed steps of lysine biosynthesis by the DAP pathway: DHDPS-catalyzed formation of unstable heterocycle by condensation of aspartate semi-aldehyde with pyruvate; and DHDPR-catalyzed reduction of the unstable heterocycle to produce tetrahydrodipicolinate, in presence of NADH. We designed a specific DHDPS mutant to be used for simple and informative screening of inhibitors on the basis of intrinsic tryptophan fluorescence change. The developed assay delivers valuable information on binding of inhibitors to allosteric site of DHDPS as a complementary analysis to kinetic studies. This also provides additional structure-activity relationships that are useful in guiding further design of inhibitors. In addition, we have observed that some of these mutations disrupt the quaternary structure of the protein. These results shed light on the mechanism and evolution of enzymatic activity of DHDPS.