Mutations in Dynamic Structural Elements Alter the Kinetics and Fidelity of the Multifunctional Class II Lanthipeptide Synthetase, HalM2

Abstract

Class II lanthipeptide synthetases (LanM enzymes) catalyze the multistep post-translational modification of genetically encoded precursor peptides into macrocyclic (often antimicrobial) lanthipeptides. The reaction sequence involves dehydration of serine/threonine residues, followed by intramolecular addition of cysteine thiols onto the nascent dehydration sites to construct thioether bridges. LanMs utilize two separate active sites in an iterative yet highly coordinated manner to maintain a remarkable level of regio-and stereochemical control over the multistep maturation. The mechanisms underlying this biosynthetic fidelity remain enigmatic. We recently demonstrated that proper function of the haloduracin β synthetase (HalM2) requires dynamic structural elements scattered across the surface of the enzyme. Here, we perform kinetic simulations, structural analysis of reaction intermediates, hydrogen−deuterium exchange mass spectrometry studies, and molecular dynamics simulations to investigate the contributions of these dynamic HalM2 structural elements to biosynthetic efficiency and fidelity. Our studies demonstrate that a large, conserved loop (HalM2 residues P349−P405) plays essential roles in defining the precursor peptide binding site, facilitating efficient peptide dehydration, and guiding the order of thioether ring formation. Moreover, mutations near the interface of the HalM2 dehydratase and cyclase domains perturb cyclization fidelity and result in aberrant thioether topologies that cannot be corrected by the wild type enzyme, suggesting an element of kinetic control in the normal cyclization sequence. Overall, this work provides the most comprehensive correlation of the structural and functional properties of a LanM enzyme reported to date and should inform mechanistic studies of the biosynthesis of other ribosomally synthesized and post-translationally modified peptide natural products.

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

“…Finally, following the formation of the second thioether ring (which represented the slowest overall step in the entire pathway), the final two thioether rings were very efficiently installed, suggesting that intermolecular interactions between HalM2 and HalA2 may be changing during maturation in a manner that facilitates more efficient docking and modification of the peptide in the cyclase active site. 42 Several constitutional isomers were distinguishable for compounds 2-6, indicating that the dehydration order is not strict, though there is a general preference for an Nto C-terminal directionality. The cyclization order was ring B  A  C/D.…”

“…We developed an HDX-MS workflow for HalM2 that reproducibly generates over 200 HalM2derived peptides, provides nearly 90% sequence coverage, and allows measurement of deuterium uptake differences as small as 0.4 Da at the 99% confidence interval (Figure 8A). [40][41][42] The most dynamic regions of the free enzyme (e.g. highest deuterium uptake) include the Nterminal capping helices, the interface between the kinase activation domain and the activation loop, the interface between the cyclase domain and the dehydrase C-lobe, and a large loop spanning HalM2 residues P349-P405 (Figure 8B).…”

“…The sequence corresponding to H110-P123 is not ordered in the CylM crystal structure. 40,42,63 Replacement of segments of the H110-P123 region with unstructured Gly-Ser linkers strongly reduced the HalA2 binding affinity (Figure 10A). 40 Moreover, photochemical crosslinking studies suggested direct contact between the HalA2 leader peptide and the E39-L50 element.…”

“…In Section 3, we briefly introduce the haloduracin  synthetase (HalM2) and its substrate peptide (HalA2) from Bacillus halodurans as a model system for elucidating structure-function relationships in class II lanthipeptide synthetases. Sections 4-6 highlight our systematic and detailed investigations into the HalM2:HalA2 system, 38,[40][41][42][43] and illustrate the many powerful and unique advantages of high-resolution mass spectrometry (MS) for characterizing the mechanisms of lanthipeptide biosynthesis. Namely, MS-based methods can assess every level of protein/peptide structure -all of which are relevant to understanding the mechanisms of lanthipeptide biosynthesis.…”

Control of Nucleophile Chemoselectivity in Cyanobactin YcaO Heterocyclases PatD and TruD

“…Dynamic regions are also observed in other RiPP enzymes such as the lanthionine synthetases ProcM and HalM, in the regions that control substrate access. 35,36 In recent years, promiscuous heterocyclases including PatD and LynD have been widely used in synthesis of diverse compounds. This work has been facilitated by fusion of RSI to activate the enzymes so that long precursor peptides are not needed.…”