Defining the functional properties of cyclopropane fatty acid synthase from Pseudomonas aeruginosa PAO1.

Cyclopropane fatty acid synthases (CFAS) catalyze the conversion of unsaturated fatty acids to cyclopropane fatty acids (CFAs) within bacterial membranes. This modification alters the biophysical properties of membranes and has been correlated with virulence in several human pathogens. Despite the central role played by CFAS enzymes in regulating bacterial stress responses, the mechanistic properties of the CFAS enzyme family and the consequences of CFA biosynthesis remain largely uncharacterized in most bacteria. We report the first characterization of the CFAS enzyme from Pseudomonas aeruginosa (PA), an opportunistic human pathogen with complex membrane biology that is frequently associated with antimicrobial resistance and high tolerance to various external stressors. We demonstrate that CFAs are produced by a single enzyme in PA and that cfas gene expression is upregulated during the transition to stationary phase and in response to oxidative stress. Analysis of PA lipid extracts reveal a massive increase in CFA production as PA cells enter stationary phase and help define the optimal membrane composition for in vitro assays. The purified PA-CFAS enzyme forms a stable homodimer and preferentially modifies phosphatidylglycerol lipid substrates and membranes with a higher content of unsaturated acyl chains. Bioinformatic analysis across bacterial phyla shows highly divergent amino acid sequences within the lipid-binding domain of CFAS enzymes, perhaps suggesting distinct membrane-binding properties among different orthologs. This work lays an important foundation for further characterization of CFAS in P. aeruginosa and for examining the functional differences between CFAS enzymes from different bacteria.

Exploring the Heterogeneous Structural Dynamics of Class II Lanthipeptide Synthetases with Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS).

Class II lanthipeptide synthetases (LanM enzymes) catalyze the installation of multiple thioether bridges into genetically encoded peptides to produce macrocyclic lanthipeptides, a class of biologically active natural products. Collectively, LanM enzymes install thioether rings of different sizes, topologies, and stereochemistry into a vast array of different LanA precursor peptide sequences. The factors that govern the outcome of the LanM-catalyzed reaction cascade are not fully characterized but are thought to involve both intermolecular interactions and intramolecular conformational changes in the [LanM:LanA] Michaelis complex. To test this hypothesis, we have combined AlphaFold modeling with hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of a small collection of divergent LanM/LanA systems to investigate the similarities and differences in their conformational dynamic properties. Our data indicate that LanA precursor peptide binding triggers relatively conserved changes in the structural dynamics of the LanM dehydratase domain, supporting the existence of a similar leader peptide binding mode across the LanM family. In contrast, changes induced in the dynamics of the LanM cyclase domain were more highly variable between enzymes, perhaps reflecting different peptide-cyclase interactions and/or different modes of allosteric activation in class II lanthipeptide biosynthesis. Our analysis highlights the ability of the emerging AlphaFold platform to predict protein-peptide interactions that are supported by other lines of experimental evidence. The combination of AlphaFold modeling with HDX-MS analysis should emerge as a useful approach for investigating other conformationally dynamic enzymes involved in peptide natural product biosynthesis.