Structure-function Study of PchB, an Isochorismate-Pyruvate Lyase from <italic>Pseudomonas aeruginosa</italic>

Abstract

Enzymes act as biological catalysts for chemical reactions that would otherwise occur at rates insufficient for the survival and growth of an organism. Understanding the fundamental forces that drive catalysis in enzymes is applicable in the production of novel antibiotics through rational drug design and advancing our understanding of how enzymes achieve incredible rate enhancements over their uncatalyzed reactions. A body of work to understand these forces has been established in the chorismate mutases and the pericyclic reaction they catalyze in the production of prephenate from chorismate. In Pseudomonas aeruginosa a structural homologue of the Escherichia coli chorismate mutase EcCM has been found in the isochorismate-pyruvate lyase PchB. While the physiological role of PchB is that of an isochorismate-pyruvate lyase (IPL) in the production of salicylate and pyruvate from isochorismate, PchB also exhibits an adventitious chorismate mutase (CM) activity. The contribution of forces that drive IPL and CM activity in PchB is somewhat controversial with differing hypothesises of transition state stabilization through electrostatic interactions and the formation of a reactive substrate through conformational destabilization. Crystallization and mutational studies have shown the importance of a positive charge at position 42 for efficient catalysis within a dynamic active site loop region of PchB. The charge swap PchB mutant K42E showed no detectable activity and PchB mutant K42A retaining one percent of WT activity. The WT PchB and PchB mutant K42E crystal structures with the products of the IPL activity (salicylate and pyruvate) bound in the active site show a conserved active site architecture among each other and that of previously solved PchB mutant K42A structure with salicylate and pyruvate and WT PchB structure with two pyruvate molecules bound in the active site. The conservation of the active site architecture strongly suggests that the differences in the catalytic activities are due to the change in chemical nature of the residue at position 42 within the dynamic active site loop region. The initial steps into the exploration of the contributions of the dynamic active site loop region to IPL and CM activity in PchB through the current nuclear magnetic resonance spectroscopy assignment of 32 of the 101 backbone resonances of PchB set the foundation for further assignment and collection of relaxation data that will provide loop movement analysis on the picosecond to millisecond time scale.