In Vitro Biosynthesis of the Prepeptide of Type‐III Lantibiotic Labyrinthopeptin A2 Including Formation of a CC Bond as a Post‐Translational Modification

Abstract

Lantibiotics are ribosomally synthesized peptide antibiotics containing the amino acids lanthionine (Lan) and methyllanthione (MeLan) as the most important and characteristic post-translational modification.[1] Work on the in vitro characterization of the enzymatic processing of lantibiotics, in particular for the formation of lanthionine, was described in reports on lacticin 481 [2] and haloduracin.[3] Recently, we identified a new family of lantibiotics, named labyrinthopeptins, which are produced by the actinomycete Actinomadura namibiensis DSM 6313. [4][5][6] The structure elucidation of labyrinthopeptin A2 (Scheme 1) mainly performed by X-ray crystallography highlighted a new amino acid, named labionin (Lab) (Scheme 1). Labionin is a triamino acid with a 2S,4S,8R configuration, and consists of a central quarternary carbon atom with a lanthionine motif and an unusual methylene bridge, which establishes a covalent link to a further amino acid moiety. This structure facilitates the formation of two rings in a linear peptide. Bioactivity assays revealed that labyrinthopeptin A2 has an excellent efficacy against neuropathic pain in an in vivo mouse model (ED 50 = 50 mg kg À1 ). The additionally found labyrinthopeptins A1 and A3 have been proposed as analogues of the A2 structure containing different amino acid constituents and an alternated ring size of the lanthionine motif.[4]The overall identification and sequencing of the gene cluster revealed only five genes which could be assigned to the biosynthesis of labyrinthopeptins. These contained two structural genes (labA1/A2) as precursors of labyrinthopeptin A1/ A3 and A2, two genes with sequence similarity to ATPdependent ABC transporters for the putative peptide export (labT1 and labT2), and one gene (labKC) for a putative modifying enzyme.[4] LabKC (MW = 95 kDa) is a two-domain protein consisting of an N-terminally conserved domain with features of an eukaryotic Ser/Thr protein kinase and a C-terminal domain with low sequence homology to LanC cyclases. In comparison to LanC cyclases, important active site residues, such as the zinc binding motif identified for the nisin cyclase, are missing in LabKC. [7] Moreover, the amino acid sequence of LabKC shows high homology to the SapB modifying enzyme RamC and to sequences from gene clusters of a considerable number of sequenced actinomycete strains (Supporting Information). [4,8] Therefore, we assign labyrinthopeptins as type-III lantibiotics.Herein we present the first in vitro reconstitution of the pre-labyrinthopeptin A2 biosynthesis in which a C À C bond formation is catalyzed by LabKC. This reaction involves the unprecedented requirement of guanosine triphosphate (GTP) for the phosphorylation and dehydratation reaction of serines. In other in vitro syntheses of lantibiotics adenosine triphosphate (ATP) and not GTP is used. [2,3] From the obtained data a biosynthetic model has been deduced. To our knowledge, this is the first time that GTP has been shown to act as a cosubstrate in the formation of lanthionine-ty...

In vitro activity of the nisin dehydratase NisB

“…The leader peptide binding site has been previously shown to not be located in this C-terminal domain (25). The proposed activation mechanism for LanB dehydratases stands in contrast to the activation of the hydroxyl groups of Ser and Thr by phosphorylation during the dehydration process of classes II, III, and IV lanthipeptides (6,7,29). Why glutamylation of Ser/Thr would evolve as an alternative means of Ser/Thr activation is unclear.…”

Expanded Natural Product Diversity Revealed by Analysis of Lanthipeptide-Like Gene Clusters in Actinobacteria

“…Class II enzymes are generically named LanMs, which are single polypeptides containing an N-terminal dehydratase domain that bears no homology to any functionally known enzymes and a C-terminal LanC-like cyclase domain (13,14). Class III and class IV synthetases are trifunctional enzymes termed LanKC (15) and LanL (16), respectively. These enzymes contain an N-terminal lyase domain and a central kinase domain but differ in their C termini.…”

Evolution of lanthipeptide synthetases

“…1A). In some cases, this reaction is coupled with a second Michael-type addition of the resulting enolate to a second dehydroalanine to produce a labionin structure (10). Genetic and biochemical studies have revealed four distinct classes of lanthipeptides according to their biosynthetic machinery (7,11,12) (Fig.…”

“…For class II lanthipeptides, the reactions are carried out by a single lanthipeptide synthetase, LanM, containing an N-terminal dehydratase domain that bears no homology to LanB, and a C-terminal LanC-like cyclase domain. Class III and class IV lanthipeptides are synthesized by trifunctional enzymes LanKC (10,13) and LanL (12), respectively. These enzymes contain an N-terminal lyase domain and a central kinase domain but differ in their C termini.…”