Discovery of unique lanthionine synthetases reveals new mechanistic and evolutionary insights

Abstract

Lantibiotic synthetases are remarkable biocatalysts generating conformationally constrained peptides with a variety of biological activities by repeatedly utilizing two simple posttranslational modification reactions: dehydration of Ser/Thr residues and intramolecular addition of Cys thiols to the resulting dehydro amino acids. Since previously reported lantibiotic synthetases show no apparent homology with any other known protein families, the molecular mechanisms and evolutionary origin of these enzymes are unknown. In this study, we present a novel class of lanthionine synthetases, termed LanL, that consist of three distinct catalytic domains and demonstrate in vitro enzyme activity of a family member from Streptomyces venezuelae. Analysis of individually expressed and purified domains shows that LanL enzymes install dehydroamino acids via phosphorylation of Ser/Thr residues by a protein kinase domain and subsequent elimination of the phosphate by a phosphoSer/Thr lyase domain. The latter has sequence homology with the phosphothreonine lyases found in various pathogenic bacteria that inactivate host mitogen activated protein kinases. A LanC-like cyclase domain then catalyzes the addition of Cys residues to the dehydro amino acids to form the characteristic thioether rings. We propose that LanL enzymes have evolved from stand-alone protein Ser/Thr kinases, phosphoSer/Thr lyases, and enzymes catalyzing thiol alkylation. We also demonstrate that the genes for all three pathways to lanthionine-containing peptides are widespread in Nature. Given the remarkable efficiency of formation of lanthionine-containing polycyclic peptides and the latter's high degree of specificity for their cognate cellular targets, it is perhaps not surprising that (at least) three distinct families of polypeptide sequences have evolved to access this structurally and functionally diverse class of compounds.

Figure 1. Putative lantibiotic biosynthesis in S. venezuelae.

(A) Posttranslational modification of precursor peptides by lantibiotic synthetases. Following ribosomal synthesis of the precursor peptides, LanB or LanM enzymes dehydrate Ser and Thr to afford Dha and Dhb, respectively. Subsequently, LanC or LanM catalyze intramolecular addition of Cys thiols onto the dehydro amino acids in a stereo- and regio-selective manner to form Lan and MeLan. (B) The biosynthetic gene cluster of S. venezuelae consists of the synthetase gene venL, the precursor gene venA, and two components of an ABC transporter venT and venH, encoding the ATP-binding and permease subunits, respectively. (C) A conserved domain search for VenL identified a putative protein kinase domain and LanC-like domain shown in blue and red, respectively. Location of conserved residues in the domains is shown as colored boxes. (D) Primary sequence of VenA. The cysteine residues and possible dehydration sites are highlighted in red and blue, respectively. The putative core region is underlined.

Figure 2. MALDI-ToF MS analysis of in vitro enzyme assays of His₆-VenA with VenL and VenL deletion proteins.

Mass spectra are depicted of (A) His₆-VenA, (B) His₆-VenA after incubation with His₆-VenL, (C) His₆-VenA after incubation with His₆-VenL-ΔC, (D) His₆-VenA after incubation with His₆-VenL-ΔLC, and (E) His₆-VenA after incubation with both His₆-VenL-ΔLC and His₆-VenL-ΔKC. The assignments of observed peaks are shown in the spectra, in which SM indicates the starting material (His₆-VenA).

Figure 3. Sequence alignment of LanL protein family members with other proteins.

(A) Alignment of the N-terminal regions of the LanL family with RamC, AmfT and three protein kinases (PK-1 from S. clavuligerus ATCC 27064, YP_002190269; PksC from Streptomyces coelicolor A3(2), NP_628010; and PKA from mouse, NP_032880). Fully and partially conserved residues are highlighted in yellow and green, respectively, with cyan indicating similar amino acids. Roman numerals refer to subdomains conserved across the Ser/Thr protein kinase family. Blue stars indicate the amino acids that are highly conserved in protein kinases. (B) The N-terminal regions of the LanL family, which exhibit no homology with kinases, were aligned with RamC family (RamC and AmfT) and OspF family (OspF, SpvC, and HopAI1) members. Color coding as in (A). Red stars indicate catalytic residues in the proposed mechanism of SpvC that are conserved in the LanL family, and black stars indicate proposed catalytic residues that are not found in LanL proteins. For full sequences and accession codes, see Figures S6 and S8.

Figure 4. Determination of the structure of venezuelin.

Red arrows indicate the b and y″ ions observed in ESI tandem mass spectra of the products of incubation with His₆-VenL followed by treatment with GluC. The observed fragmentation pattern is shown for (A) VenL-modified VenA, (B) VenL-modified VenA-C34A, (C) VenL-modified VenA-C45A, (D) VenL-modified VenA-C32A, and (E) VenL-modified VenA-C50A. Dehydrated amino acids and cysteine residues in each analog are shown in blue and green, respectively. See Figure S3 for the MS/MS spectra. (F) The topology of (methyl)lanthionines in venezuelin. Magenta arrows indicate the position and direction of Lan/MeLan ring formation.

Figure 5. Posttranslational modification by LanL enzymes.

(A) Illustration of the three catalytic domains constituting LanL enzymes. The positions of conserved residues important for catalysis are shown in darker colors. (B) Scheme of post-translational modification by LanL enzymes to install Lan/MeLan into the substrate LanA peptides. R = H or Me.

Figure 6. Phylogenetic tree of 16S rRNA of species having putative lantibiotic synthetase genes.

Species with lanL, lanB, and lanM genes are highlighted in yellow, green, and magenta, respectively. The phylogenetic tree was constructed based on the Ribosomal Database Project . For the bacterial strains used and gene accession numbers, see Tables S3 and S4.