BPS2026 – Redox control of phenazine biosynthesis via disulfide bond engineering of the PhzM methyltransferase

Bidirectional information exchange is fundamental to communication between distinct systems. Biological and electronic frameworks generally employ different modalities, but both use oxidation and reduction (redox) for signaling. Electronically regulating the biosynthesis of signaling molecules through control of electron transfer enables external modulation of enzyme activity. The phenazine class of multifunctional redox-active compounds are key contextual signals in microbial interactions, for example, regulating biofilm synthesis. Examination of the biosynthesis pathway for pyocyanin (PYO), a major phenazine produced by Pseudomonas aeruginosa , identified the PhzM methyltransferase as an appropriate nexus for engineering redox-mediated control. PhzM uses SAM to methylate phenazine-1-carboxylic acid (PCA), ultimately directing the phenazine pathway toward PYO. Little information is available about the PhzM active site, so bioinformatic analyses and alanine scanning mutagenesis were employed to identify residues critical for function. The results guided protein engineering intended to control enzymatic activity by introducing a redox-active disulfide bond into PhzM at a position disrupting the active site. We screened 30 variants predicted to form different single disulfide bonds, hoping to identify enzymes that are active when reduced and inactive when oxidized. Several variants showed the desired decrease in activity when expressed in the oxidizing cytoplasm of NEB Shuffle cells; one variant, surprisingly, showed increased activity. To further understand the effects of the putative disulfides, five PhzM variants were purified and their ordered bi-bi kinetics were compared to wild type. Assays included the PhzS enzyme catalyzing the next step, yielding pyocyanin that was monitored by visible absorbance. This approach will allow electronic stimuli (via electrode-generated hydrogen peroxide) to modulate enzymatic activity. If successful, this proof of concept will demonstrate microelectronic control over an intracellular biosynthetic enzyme, establishing a foundation for electronically regulated biological activities.