Lanthipeptides from the Same Core Sequence: Characterization of a Class II Lanthipeptide Synthetase from Microcystis aeruginosa NIES-88

Abstract

Class II lanthipeptide synthetases (LanMs) are relatively promiscuous to core peptide variations. Previous studies have shown that different LanMs catalyze identical reactions on the same core sequence fused to their respective cognate leaders. We characterized a new LanM enzyme from Microcystis aeruginosa NIES-88, MalM, and demonstrated that MalM and ProcM exhibited disparate dehydration and cyclization patterns on identical core peptides. Our study provided new insights into the regioselectivity of LanMs and showcased an appropriate strategy for lanthipeptide structural diversity engineering.Letter pubs.acs.org/OrgLett

Investigating the Specificity of the Dehydration and Cyclization Reactions in Engineered Lanthipeptides by Synechococcal SyncM

“…Moreover, we suggest that these limitations will depend on the chosen peptide. Our results support the scenario where the substrate characteristics play an essential role in the processing of the peptide. ,,, Therefore, the structure of the core sequence can help prevent dehydration, cyclization, or both. ,, However, it is also possible that the interaction between SyncM and nonsuccessful designs could be due to unknown steric hindrances . Moreover, we do not discard the possibility that the chosen leader expressed with the different hybrids could also affect the result.…”

“… 33 , 41 , 51 However, it is also possible that the interaction between SyncM and nonsuccessful designs could be due to unknown steric hindrances. 52 Moreover, we do not discard the possibility that the chosen leader expressed with the different hybrids could also affect the result.…”

“…For the leader release, the fractions with pure full peptides were freeze-dried and resuspended in 1 mL of 100 mM Tris-HCL, pH 6. Then, purified NisP was added as indicated by Montalbán-López et al 56 (1:10 ratio) and incubated at 30 °C for 2 h. 52 For SyncA7 variants, the sample was resuspended in 50 mM Tris pH 8 and 1 mM DTT. Then, the cleavage reaction mix containing (as recommended by the authors 38 ) ∼10 μM LaTh150 protease and ∼100 μM substrate was incubated overnight at room temperature.…”

“…For the leader release, the fractions with pure full peptides were freeze-dried and resuspended in 1 mL of 100 mM Tris-HCL, pH 6. Then, purified NisP was added as indicated by Montalbán-López et al (1:10 ratio) and incubated at 30 °C for 2 h . For SyncA7 variants, the sample was resuspended in 50 mM Tris pH 8 and 1 mM DTT.…”

Facile Method for Determining Lanthipeptide Stereochemistry

“…However, the stereochemistry of the Lan and MeLan residues of the great majority of newly reported lanthipeptides is not determined. Therefore, as more and more lanthipeptides are discovered by genome mining, − a convenient method for determining their stereochemistry that is accessible to most laboratories would be valuable. We report here such a method including access to standards that use biochemical approaches that are complementary to chemical synthesis and methods available in most if not all laboratories studying lanthipeptides or RiPPs.…”