Mechanistic Investigations on Anchored Substrates and Inhibitors for Carrier Protein Dependent Acyl-Homoserine Lactone Synthases. - PROJECT SUMMARY Gram-negative bacteria count N-acyl-L-homoserine lactone (AHL) autoinducer signals to estimate local population densities through a cell-counting mechanism called quorum sensing (QS). To facilitate QS, AHL synthases recognize specific Acyl Carrier Protein (acyl-ACP) and S-adenosyl-L-methionine (SAM) substrates to generate specific AHL signals for the bacterium. Bacteria counts these AHL signals as ‘votes’ to determine if their local population has reached a sufficient quorum to collectively regulate gene expressions such as bioluminescence, and virulence. QS inhibitors are therefore useful leads as antivirulence compounds and as chemical probes to investigate social behavior in bacteria. Over the past twelve years, our group has developed a versatile experimental toolkit to investigate the mechanisms of quorum signal synthesis. Our long-term goals are to a) establish the molecular determinants that dictate fidelity in substrate recognition and AHL signal synthesis and b) explore mechanistic differences between AHL synthases and acyl-ACP utilizing enzymes to develop QS-specific inhibitors and c) evaluate the merits and demerits of ligand anchoring approaches in developing protein-free substrates and small molecule inhibitors for ACP-dependent enzymes. Enzymes that react with acyl carrier protein substrates typically involve a series of carefully orchestrated protein-protein interactions to bind and react with their cognate substrates. As a consequence, protein-free pantetheine-, CoA-based substrates demonstrate poor reactivity posing barriers to the rational design of small molecule inhibitors for ACP-dependent enzymes. To address this challenge, we recently embarked on a mechanistic study to investigate the extent of ACP-enzyme protein-protein footprint for a series of substrates bound to 3-oxoC6-ACP preferring EsaI and C8-ACP preferring BmaI1 AHL synthases. Our discovery of a significantly reduced protein-protein footprint from a single hydrogen bond anchor in EsaI has opened up exciting new prospects for developing small-molecule anchored substrates and inhibitors for ACP-dependent enzymes. Building upon this discovery, we hypothesize that increasing the number of anchors between the substrate and enzyme should further reduce the need for protein-protein footprint, enhance the catalytic efficiencies of protein-free substrates and inform the rational design of small molecule active site specific inhibitors. This MIRA proposal will take our program in a new direction by integrating chemical synthesis, structural biology, microbiology, protein biochemistry and mechanistic enzymology approaches to evaluate a previously untested ligand anchoring hypothesis on both acyl-chain cleaving (AHL synthases) and acyl-chain modifying (fatty acid biosynthesis) ACP-utilizing enzymes. Our findings will inform the rational design of pantetheine-based, active site specific inhibitors for medicinally important carrier protein dependent enzymes.
