Ammonia-utilizing MST enzymes: deciphering the mechanistic basis of reaction specificities, feedback inhibition, and allosteric interactions

• Applicant: Professor Dr. Reinhard Sterner
• Subject Area: Biochemistry ; Bioinformatics and Theoretical Biology ; Structural Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 548227467

The two evolutionarily related bi-enzyme complexes anthranilate synthase (AS) and amino-deoxychorismate synthase (ADCS) are composed of structurally and functionally similar glutaminase and synthase subunits whose catalytic activities are tightly coordinated. The synthase subunits perform chemical conversions of chorismate by nucleophilic addition of ammonia that is produced by the glutaminase subunits, resulting in distinct precursor molecules for the biosynthesis of tryptophan (AS) and folate (ADCS). Moreover, tryptophan is bound to both synthase units but with different functional consequences, acting as feedback inhibitor (AS) or as structure-stabilizing element (ADCS). It is interesting from both the enzymatic and the evolutionary point of view, to identify similarities and differences between AS and ADCS to shed light on (i) the catalytic mechanisms of the synthase reactions, (ii) the structural basis and the evolution of feedback inhibition by tryptophan, and (iii) the mechanistic basis of synthase-glutaminase inter-subunit communication. A unique opportunity to address these questions is provided by paAS and paADCS from the pathogenic organism Pseudomonas aeruginosa, because these enzymes are composed of different synthase subunits (paTrpE or paPabB) while employing the same amphibolic glutaminase subunit (paPabA). To compare the catalytic mechanisms of paTrpE and paPabB, affinities and rate constants associated with binding of the substrate chorismate, the cofactor Mg2+ and the reaction intermediate aminodeoxyisochorismate will be determined by equilibrium titrations and pre-steady-state kinetic measurements. In addition, the reaction stoichiometries of the glutaminase versus synthase reactions and the origin of the ammonia incorporated into the reaction products will be evaluated by means of steady-state assays and NMR spectroscopy. Using crystal structures of paAS and paADCS in combination with enhanced sampling MD techniques as well as ancestral sequence reconstruction, amino acid residues responsible for the different function of tryptophan in paTrpE and paPabB will be identified and used to engineer tryptophan-inhibited paPabB variants. Finally, the molecular mechanisms of inter-subunit communication will be elucidated by identifying differences in the paTrpE-paPabA versus paPabB-paPabA interfaces by a comparative structural and functional analysis.

• DFG Programme: Research Grants