Lincosamide synthetase--a unique condensation system combining elements of nonribosomal peptide synthetase and mycothiol metabolism

Abstract

In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of S. lincolnensis strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of ccbZ gene adjacent to ccbN, the counterpart of lmbN, suggesting the gene rearrangement, evident also from still active internal translation start in lmbN, and indicating the direction of lincosamide biosynthesis evolution. The in vitro test with LmbN-CP, LmbC and the newly identified S. lincolnensis phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a holo-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.

Fig 1. Structures and biosynthetic gene clusters of natural lincosamides.

The resistance genes are marked in grey, the genes homologous in both gene clusters are black. The genes highlighted by red line exhibit inactivation pattern enabling their assignment to condensation reaction, i.e. formation of amide bond (in red oval in the structures).

Fig 2. The lincomycin biosynthetic pathway.

Gray background highlights the condensation step solved herein. Red—proteins with already proved functions, A—adenylation domain, CP—carrier protein, PPL—4-propyl-L-proline, MTL—methylthiolincosamide.

Fig 3. The biological activity assay of S. lincolnensis inactivation mutants.

The inhibition zones correspond to the antibiotic production. WT—wild type strain (positive control); ΔT, ΔN, N-ΔID, N-ΔCP indicate the respective disrupted lincomycin biosynthetic gene or its part; NC—not complemented; PPL or MTL—complemented by the respective intermediate of lincomycin biosynthesis. For each sample, at least two independent cultivations were evaluated in triplicates as described in Material and methods. A typical result is shown.

Fig 4. The biological activity assay of S. lincolnensis inactivation mutants.

The inhibition zones correspond to the antibiotic production. WT—wild type strain (positive control); ΔC, ΔD, ΔE, ΔF, ΔV, ΔIH, ΔQ—indicate the respective disrupted lincomycin biosynthetic gene; NC—not complemented; PPL, MTL or PPL+MTL—indicate complementation by the respective intermediate of lincomycin biosynthesis or their combination. For each sample, at least two independent cultivations were evaluated in triplicates as described in Material and methods. A typical result is shown.

Fig 5. Evidence of two translation starts in LmbN coding gene.

(A) Western blot analysis of LmbN forms produced by S. lincolnensis, and heterologously in S. coelicolor. 1 and 7: standards of MW (His₈-tagged LmbN, His₈-tagged LmbN-ID, His₈-tagged LmbN-CP); 2: S. lincolnensis ΔlmbN mutant; 3: S. lincolnensis WT; 4: S. coelicolor M145 WT; 5: S. coelicolor M145 containing mutant form of lmbN with artificial stop codon introduced immediately downstream of the translation start 1; 6: S. coelicolor M145 containing native lmbN gene. (B) The detailed scheme of internal translation start (start 2) in lmbN. The red colour corresponds to CP domain coding sequence; the green colour corresponds to amino sugar isomerase domain (ID) coding sequence. RBS—ribosome binding site. Start 1—regular translation start producing full length LmbN.