Structure, dynamics, evolution and catalysis of phosphohexomutase from Pseudomonas aeruginosa
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] [alpha]-D phosphohexomutases are ubiquitous in the environment and participate in carbohydrate metabolism by catalyzing an intramolecular phosphoryl transfer. Biophysical and biochemical techniques have been utilized to study a phosphohexomutase from the human pathogen Pseudomonas aeruginosa, which contributes to its infectivity and virulence. The research work described herein includes use of computational analysis, mutagenesis, steady-state kinetics, hydrogen deuterium exchange by mass spectrometry (HDXMS), X-ray crystallography and small angle X-ray scattering (SAXS) to investigate important features underlying the catalytic mechanism. With mutagenesis and kinetics, a key amino acid in the active site of enzyme that is essential to function was identified. Computational analysis coupled with biochemical characterizations revealed a network of co-evolving residues at domain interface, site of conformational change. Based on HDXMS and SAXS studies, dephosphorylation of the enzyme results in a global increase in flexibility that coincides with a mandatory step of intermediate reorientation during the catalytic cycle. This study provided insight into the role of conformational plasticity in substrate binding and phosphorylation in the enzymatic reaction.
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