Mechanistic Investigations into the Catalytic Mode of a Dehydratase Complex Involved in the Biosynthesis of Lantibiotic Cacaoidin

Abstract

Comprehensive SummaryDehydration of serine/threonine residues necessitates the activity of a dehydratase enzyme (domain) during the biosynthesis of RiPP. Recently, it was reported that dehydrations in the thioviridamide pathway relies on a distinct dehydratase complex which showcases the activities of a phosphotransferase TvaC for serine/threonine phosphorylation and a lyase TvaD for subsequent phosphate elimination. Herein, we report that dehydration reactions in the pathway of lantibiotic cacaoidin involves a similar dehydratase complex, CaoK/CaoY. Remarkably, this dehydratase complex exhibits flexible enzymatic activity and tolerates significant variations in its substrate peptide sequence. By binding with the leader peptide (LP) sequence of precursor peptide CaoA, the dehydration reactions proceed directionality from the C‐terminus of the core peptide (CP) to its N‐terminus, and C‐terminally truncated variants of CP are acceptable. We show that fusing CaoK to CaoY in a 1:1 molar ratio enables the resulting enzyme CaoYK to exert enhanced dehydration activity. CaoK binds with the LP to improve its own solubility and to ensure the phosphate transfer activity, while CaoY functions in a manner independently of LP. This work advances our understanding of the dehydration process during cacaoidin formation, and provides useful enzymes and methods for the studies of the rapidly emerging RiPPs.This article is protected by copyright. All rights reserved.

Characterization of a LanC-free pathway for the formation of an ll-MeLan residue and an alloAviMeCys residue in the newly identified class V lanthipeptide triantimycins

“…We investigated Streptomyces species (taxonomy ID: 1883) in the NCBI reference Sequence database for class Va lanthipeptide candidates using the cblaster tool, 11 with the sequences of CaoK/CaoY (cases of LanK/LanY in cacaoidin) as the input. 9 To filter out false positives in which LanC cyclases or other lanthipeptide synthetases are co-existent, 7,12 the co-occurrence of CaoD/CaoX (cases of LanD/LanX in cacaoidin) was tentatively utilized as a secondary confirmation. This approach was based on the observation that all known class Va lanthipeptides exhibit the co-occurrence of LanK/LanY/LanD/LanX (Fig.…”

“…2 e ,8 A pathway-dedicated cyclase LanC (class I) or a cyclase domain (class II–IV) fused to a megasynthetase directs the thia-Michael addition of a cysteine to an intramolecular dehydroalanine (Dha) or dehydrobutyrine (Dhb) residue for the formation of Lan or MeLan residue. 8 a For class V lanthipeptides, the split kinase-like LanK and effector-like protein LanY act as the primary PTM enzymes for the formation of Dhx (Dha or Dhb) residues, 9 which are presumed to be substrates for d -amino acid(s), (Me)Lan residue(s), and the (2 S , 3 S )-AviMeCys residue (Fig. S1†).…”