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Christopher Thibodeaux

Correlating form and Function in Peptide Biosynthetic Enzymes

Thu, Sep. 8, 2022, 4:30pm
Taylor Auditorium, Frick Chemistry Lab B02
Host: Mohammad Seyedsayamdost

The reaction catalyzed by class II lanthipeptide synthetase (LanM) enzymes involves dehydration of serine/threonine residues in the LanA precursor peptide, followed by intramolecular addition of cysteine thiols to form thioether bridges.  LanM enzymes function in an iterative manner, whereby multiple thioether linkages are installed in a sequential fashion into the maturing LanA intermediate.  Collectively, LanM enzymes differ dramatically in their substrate specificities, catalytic efficiencies, and other biochemical properties.  The mechanisms underlying this functional heterogeneity remain enigmatic.  This talk highlights recent efforts by our lab to untangle the complex relationships between structural dynamics, conformational changes, and biochemical function in LanM enzymes.  Using the haloduracin  synthetase (HalM2) as a model LanM system, mass spectrometry-based kinetic studies show that conformational changes and enzyme:peptide interactions influence the kinetics and sequence of biosynthetic events.  Hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies show that HalM2 is a dynamic enzyme that undergoes local structural rearrangements and long-range allosteric signalling upon peptide or nucleotide binding.  Mutations to previously overlooked, dynamic HalM2 structural elements impact the catalytic activities (dehydration and cyclization) of HalM2, providing evidence that conformational changes and enzyme-peptide interactions contribute to function.  Finally, native mass spectrometry coupled to ion mobility shows that HalM2 exists in multiple three-dimensional conformations, and that this conformational landscape is influenced by HalA2 precursor peptide binding and by post-translational modification of the HalA2 core peptide.  Cumulatively, our studies illustrate the many benefits provided by mass spectrometry for characterizing the mechanistic and structural properties of peptide biosynthetic enzymes.