Biochemical and biophysical investigation of the HalM2 lanthipeptide synthetase using mass spectrometry

Abstract

The rapid emergence of antimicrobial resistance in clinical settings has called for renewed efforts to discover and develop new antimicrobial compounds. Lanthipeptides present a promising, genetically-encoded molecular scaffold for the engineering of structurally complex, biologically active peptides. These peptide natural products are constructed by enzymes (lanthipeptide synthetases) with relaxed substrate specificity that iteratively modify the precursor lanthipeptide to generate structures with defined sets of thioether macrocycles. The mechanistic features that guide the maturation of lanthipeptides into their proper, fully-modified forms are obscured by the complexity of the multistep maturation, and the large size and dynamic structures of the synthetases and precursor peptides. Over the past several years, our lab has been developing a suite of mass spectrometry-based techniques that are ideally suited to untangling the complex reaction sequences and molecular interactions that define lanthipeptide biosynthesis. This review focuses on our development and application of these MS-based techniques to investigate the biochemical, kinetic, and biophysical properties of the haloduracin  class II lanthipeptide synthetase, HalM2.

Partially Modified Peptide Intermediates in Lanthipeptide Biosynthesis Alter the Structure and Dynamics of a Lanthipeptide Synthetase

“…As a step toward realizing this goal, we have focused on adapting structural MS techniques to advance our understanding of HalM2 and to illustrate the many benefits of MSbased approaches for investigating the structures and intermolecular interactions of RiPP biosynthetic enzymes. 48 These benefits include the ability of MS to (1) characterize the entire hierarchy of the peptide/protein structure, (2) investigate precursor peptide binding stoichiometry, (3) localize peptide binding sites, and (4) characterize intramolecular conformational changes and allosteric activation in the synthetase. Moreover, the MS methods have high sensitivity and are not always critically reliant on high-purity samples because of the high resolving power of modern mass spectrometers.…”

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

“…To begin to probe the potential origins of LanM functional heterogeneity, we have initiated detailed biophysical and biochemical studies of the haloduracin-β synthetase (HalM2) from Alkalihalobacillus halodurans, , which catalyzes seven dehydrations and installs four thioether rings into its precursor peptide, HalA2. − ,,, Currently, the only LanM enzyme for which a high-resolution structure has been solved is the cytolysin synthetase, CylM, from Enterococcus faecalis . In the AlphaFold model of HalM2 presented in Figure , each of the highlighted elements is structurally dynamic and plays a critical role in the biochemical functions of HalM2. ,, To summarize briefly, loop regions within the N-terminal capping domain function synergistically with the PP 50 -loop and kinase activation (KA) domain to form the primary binding site for the HalA2 leader peptide.…”