Julian Pac Yurrita | 2025 I.S Symposium
Name: Julian Pac Yurrita
Title: Deciphering Pyocyanin Biosynthesis: Investigating PhzS/PhzM Enzyme Dynamics and Substrate Interactions
Major: Biochemistry & Molecular Biology
Advisors: Mark J. Snider
Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is a leading cause of hospital-acquired infections, particularly in immunocompromised individuals. A major contributor to its virulence is pyocyanin, a redox-active toxin that impairs host immune function, promotes oxidative damage, and enhances biofilm formation. Despite its biological importance, the precise sequence of steps leading to pyocyanin biosynthesis remains unclear. In particular, how the two final enzymes—PhzM, a SAM-dependent methyltransferase, and PhzS, a flavin-dependent monooxygenase—coordinate the transformation of the common precursor phenazine-1-carboxylic acid (PCA) into pyocyanin has remained unresolved. Two competing pathways have been proposed: one involving hydroxylation of PCA by PhzS to form 1-hydroxyphenazine (1-HP) followed by methylation by PhzM, and the other involving initial methylation to 5-methylphenazine-1-carboxylic acid (5-MPCA) followed by hydroxylation.
To clarify this mechanism, we used fluorescence spectroscopy, titration analysis, and HPLC to evaluate substrate binding and intermediate formation. When tested individually, both PhzS and PhzM exhibited weak intrinsic binding affinities for PCA and their respective cofactors (NADH and SAM). However, when both enzymes were present in solution, fluorescence quenching experiments showed enhanced binding, consistent with cooperative complex formation. Sequential fluorescence assays further supported a hydroxylation-first pathway, with fluorescence recovery occurring only after SAM addition following PhzS activity. HPLC analysis revealed no stable accumulation of 1-HP or 5-MPCA, suggesting either rapid turnover or degradation of these intermediates. Collectively, the data support a model in which PhzS and PhzM function cooperatively, with 1-HP as the likely key intermediate in pyocyanin biosynthesis.
Posted in Symposium 2025 on May 1, 2025.
