Biosynthesis of lanthionine-constrained agonists of G protein-coupled receptors

Moll, Gert N.; Kuipers, Anneke; Rink, Rick; Bosma, Tjibbe; Vries, Louwe de; Namsolleck, Pawel

doi:10.1042/bst20200427

Cited by 5 publications

(6 citation statements)

References 66 publications

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“…After these modifications, dedicated proteases/transporters perform leader peptide proteolysis and export the mature product. Lanthipeptide synthetases demonstrate relaxed substrate specificity as they often tolerate changes made to the core peptide sequences, − a feature that has been extensively applied to bioengineer lanthipeptides. − …”

Section: Introductionmentioning

confidence: 99%

Substrate Sequence Controls Regioselectivity of Lanthionine Formation by ProcM

Fouque

Santos-Fernandez

et al. 2021

J. Am. Chem. Soc.

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Lanthipeptides belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs). The (methyl)lanthionine cross-links characteristic to lanthipeptides are essential for their stability and bioactivities. In most bacteria, lanthipeptides are maturated from single precursor peptides encoded in the corresponding biosynthetic gene clusters. However, cyanobacteria engage in combinatorial biosynthesis and encode as many as 80 substrate peptides with highly diverse sequences that are modified by a single lanthionine synthetase into lanthipeptides of different lengths and ring patterns. It is puzzling how a single enzyme could exert control over the cyclization processes of such a wide range of substrates. Here, we used a library of ProcA3.3 precursor peptide variants and show that it is not the enzyme ProcM but rather its substrate sequences that determine the regioselectivity of lanthionine formation. We also demonstrate the utility of trapped ion mobility spectrometry–tandem mass spectrometry (TIMS-MS/MS) as a fast and convenient method to efficiently separate lanthipeptide constitutional isomers, particularly in cases where the isomers cannot be resolved by conventional liquid chromatography. Our data allowed identification of factors that are important for the cyclization outcome, but also showed that there are no easily identifiable predictive rules for all sequences. Our findings provide a platform for future deep learning approaches to allow such prediction of ring patterns of products of combinatorial biosynthesis.

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Section: Introductionmentioning

confidence: 99%

Substrate Sequence Controls Regioselectivity of Lanthionine Formation by ProcM

Fouque

Santos-Fernandez

et al. 2021

J. Am. Chem. Soc.

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“… 10 , 18 This characteristic enhances their potential as an alternative system for lanthipeptide bioengineering and production, and therefore, for the future design and heterologous expression of new and potent natural or close-to-nature lanthipeptides with improved or new biological or technological activities. 2 , 8 , 13 , 16 , 19 , 20 The first ProcM-like enzyme described was identified in the Gram-negative marine cyanobacterium Prochlorococcus MIT9313. 18 This enzyme is able to process up to 29 different substrates, which are named prochlorosins, a group of unusually diverse lanthipeptides 21 found in marine cyanobacterial strains, and with unknown function.…”

Section: Introductionmentioning

confidence: 99%

“…Interestingly, this machinery can be used for the heterologous expression of different LanA substrates, with a special focus on lantibiotics, , a class of lanthipeptides characterized by the presence of antimicrobial activity, although also lanthipeptides with antifungal, antiviral, morphogenetic, anticonceptive, or antiallodynic functions have been described . Moreover, the lanthipeptide biosynthetic machinery has been used to design new-to-nature lanthipeptides, generating peptides with enhanced activity or stability. , The heterologous expression of lanthipeptides using the nisin biosynthetic machinery has been broadly used for the heterologous expression of newly designed antimicrobials. ,− However, the substrate specificity of the synthetase enzymes NisB and NisC limits the production of this type of variant compounds on a large scale. Therefore, new and promiscuous lanthipeptide synthetase enzymes with enhanced properties to fully process a large number of unrelated LanA substrates or with the ability to introduce rings with different topologies could be used for the effective heterologous production of novel bioactive lanthipeptides, either engineered variants or lanthipeptides from different (exotic) origins.…”

Section: Introductionmentioning

confidence: 99%

“…In this regard, ProcM-like enzymes constitute a unique class II lanthipeptide modification enzyme family and SyncM is a new example in this group. As a class II lanthipeptide synthetase enzyme, SyncM presents two domains, the N-terminal ATP-dependent dehydratase domain and C-terminal cyclase domain characterized by a very relaxed substrate specificity. , This characteristic enhances their potential as an alternative system for lanthipeptide bioengineering and production, and therefore, for the future design and heterologous expression of new and potent natural or close-to-nature lanthipeptides with improved or new biological or technological activities. ,,,,, The first ProcM-like enzyme described was identified in the Gram-negative marine cyanobacterium Prochlorococcus MIT9313 . This enzyme is able to process up to 29 different substrates, which are named prochlorosins, a group of unusually diverse lanthipeptides found in marine cyanobacterial strains, and with unknown function. − …”

Section: Introductionmentioning

confidence: 99%

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Functional Expression and Characterization of the Highly Promiscuous Lanthipeptide Synthetase SyncM, Enabling the Production of Lanthipeptides with a Broad Range of Ring Topologies

et al. 2021

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Lanthipeptides are ribosomally synthesized and post-translationally modified peptides characterized by the presence of lanthionine rings that provide stability and functionality. Genome mining techniques have shown their huge diversity and potential for the discovery of novel active molecules. However, in many cases, they are not easily produced under laboratory conditions. The heterologous expression of these molecules using well-characterized lanthipeptide biosynthetic enzymes is rising as an alternative system for the design and production of new lanthipeptides with biotechnological or clinical properties. Nevertheless, the substrate-enzyme specificity limits the complete modification of the desired peptides and hence, their full stability and/or biological activity. New low substrate-selective biosynthetic enzymes are therefore necessary for the heterologous production of new-to-nature peptides. Here, we have identified, cloned, and heterologously expressed in Lactococcus lactis the most promiscuous lanthipeptide synthetase described to date, i.e., SyncM from the marine cyanobacteria Synechococcus MITS9509. We have characterized the functionality of SyncM by the successful expression of 15 out of 18 different SyncA substrates, subsequently determining the dehydration and cyclization processes in six representatives of them. This characterization highlights the very relaxed substrate specificity of SyncM toward its precursors and the ability to catalyze the formation of exceptionally large rings in a variety of topologies. Our results suggest that SyncM could be an attractive enzyme to design and produce a wide variety of new-to-nature lanthipeptides with a broad range of ring topologies.

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“…[17][18][19][20] This feature has been widely utilized for bioengineering of novel lanthipeptides. [21][22][23][24][25][26][27][28] A remarkable example of substrate tolerance is the class II lanthipeptide synthetase ProcM, which was discovered in the marine picocyanobacterium Prochlorococcus MIT9313. 29 Unlike most other lanthipeptide biosynthetic pathways, the ProcM biosynthetic gene cluster does not encode just a single precursor peptide with one modifying enzyme.…”

Section: Introductionmentioning

confidence: 99%

Sequence Controlled Secondary Structure Determines Site-selectivity of Lanthipeptides

Desormeaux

et al. 2022

Preprint

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Lanthipeptides are ribosomally synthesized and post-translationally modified pep- tides that are generated from precursor peptides through a dehydration and cycliza- tion process in the biosynthetic pathways. In contrast to most other lanthipeptide synthetases, ProcM, a class II lanthipeptide synthetase, demonstrates high substrate tolerance. It is enigmatic that a single enzyme can catalyze the cyclization process of a diverse range of substrates with high fidelity. Previous studies suggested that the site- selectivity of lanthionine formation is determined by substrate sequence rather than by the enzyme. However, exactly how substrate sequence contributes to site-selective lanthipeptide biosynthesis is not clear. In this study, we performed molecular dynamic simulations for ProcA3.3 core peptide variants to explore how the predicted solution structure of the substrate without enzyme correlates to final product formation. Our simulation results support a model in which the secondary structure of the core peptide controls the ring pattern of the final product. We also demonstrate that the dehydra- tion step in the biosynthesis pathway does not influence the site-selectivity of ring formation. In addition, we performed simulation for the core peptides of ProcA1.1 and 2.8, which are well-suited candidates to investigate the connection between order of ring formation and solution structure. Simulation results indicate that in both cases, C-terminal ring formation is more likely which was supported by experimental results. Our findings indicate that the substrate sequence and its solution structure can be used to predict the site-selectivity and order of ring formation, and that secondary structure is a crucial factor influencing the site-selectivity. Taken together, these findings will fa- cilitate our understanding of the lanthipeptide biosynthetic mechanism and accelerate bioengineering efforts for lanthipeptide-derived products.

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Biosynthesis of lanthionine-constrained agonists of G protein-coupled receptors

Cited by 5 publications

References 66 publications

Substrate Sequence Controls Regioselectivity of Lanthionine Formation by ProcM

Substrate Sequence Controls Regioselectivity of Lanthionine Formation by ProcM

Functional Expression and Characterization of the Highly Promiscuous Lanthipeptide Synthetase SyncM, Enabling the Production of Lanthipeptides with a Broad Range of Ring Topologies

Sequence Controlled Secondary Structure Determines Site-selectivity of Lanthipeptides

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