Evolution-guided adaptation of an adenylation domain substrate specificity to an unusual amino acid.

Adenylation domains CcbC and LmbC control the specific incorporation of amino acid precursors in the biosynthesis of lincosamide antibiotics celesticetin and lincomycin. Both proteins originate from a common L-proline-specific ancestor, but LmbC was evolutionary adapted to use an unusual substrate, (2S,4R)-4-propyl-proline (PPL). Using site-directed mutagenesis of the LmbC substrate binding pocket and an ATP-[32P]PPi exchange assay, three residues, G308, A207 and L246, were identified as crucial for the PPL activation, presumably forming together a channel of a proper size, shape and hydrophobicity to accommodate the propyl side chain of PPL. Subsequently, we experimentally simulated the molecular evolution leading from L-proline-specific substrate binding pocket to the PPL-specific LmbC. The mere change of three amino acid residues in originally strictly L-proline-specific CcbC switched its substrate specificity to prefer PPL and even synthetic alkyl-L-proline derivatives with prolonged side chain. This is the first time that such a comparative study provided an evidence of the evolutionary relevant adaptation of the adenylation domain substrate binding pocket to a new sterically different substrate by a few point mutations. The herein experimentally simulated rearrangement of the substrate binding pocket seems to be the general principle of the de novo genesis of adenylation domains' unusual substrate specificities. However, to keep the overall natural catalytic efficiency of the enzyme, a more comprehensive rearrangement of the whole protein would probably be employed within natural evolution process

K_m values of CcbC, CcbC mutants and LmbC in reaction with various substrates.

<p>K_m values of CcbC, CcbC mutants and LmbC in reaction with various substrates.</p

Kinetic parameters of LmbC and LmbC single mutants for PPL and L-proline substrates.

<p>Kinetic parameters of LmbC and LmbC single mutants for PPL and L-proline substrates.</p

Comparison of the nonribosomal codes of CcbC and LmbC substrate binding pockets (SBPs).

<p><b>A)</b> Structures of lincomycin and celesticetin. Amino acid precursors activated by adenylation domains (A-domains) are indicated in green. <b>B)</b> Pattern of eight variable amino acid residues of CcbC and LmbC nonribosomal codes. The highly conserved D and K residues at the boundaries of the nonribosomal codes are omitted. Amino acid residues are numbered according to CcbC (first row) and LmbC (last row). The consensus code of the stand-alone L-proline-specific A-domains is shown in the middle row [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189684#pone.0189684.ref003" target="_blank">3</a>]. The residues in LmbC and CcbC SBPs, which correspond to the consensus, are underlined. Colours correspond to the individual amino acid residues in the model of CcbC/LmbC SBPs (C). <b>C)</b> Homology models of the CcbC SBP with L-proline and the LmbC SBP with PPL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189684#pone.0189684.ref003" target="_blank">3</a>].</p

Kinetic parameters of LmbC, CcbC and selected CcbC mutants for various substrates.

<p>Kinetic parameters of LmbC, CcbC and selected CcbC mutants for various substrates.</p

Diversity of Alkylproline Moieties in Pyrrolobenzodiazepines Arises from Postcondensation Modifications of a Unified Building Block

Anticancer pyrrolobenzodiazepines (PBDs) are one of several groups of natural products that contain unusual 4-alkyl-l-proline derivatives (APDs) in their structure. APD moieties of PBDs are characterized by high structural diversity achieved through unknown biosynthetic machinery. Based on LC-MS analysis of culture broths, feeding experiments, and protein assays, we show that APDs are not incorporated into PBDs in their final form as was previously hypothesized. Instead, a uniform building block, 4-propylidene-l-proline or 4-ethylidene-l-proline, enters the condensation reaction. The subsequent postcondensation steps are initiated by the introduction of an additional double bond catalyzed by a FAD-dependent oxidoreductase, which we demonstrated with Orf7 from anthramycin biosynthesis. The resulting double bond arrangement presumably represents a prerequisite for further modifications of the APD moieties. Our study gives general insight into the diversification of APD moieties of natural PBDs and provides proof-of-principle for precursor directed and combinatorial biosynthesis of new PBD-based antitumor compounds

Lincosamide Synthetase—A Unique Condensation System Combining Elements of Nonribosomal Peptide Synthetase and Mycothiol Metabolism

<div><p>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 <i>S. lincolnensis</i> 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 <i>ccbZ</i> gene adjacent to <i>ccbN</i>, the counterpart of <i>lmbN</i>, suggesting the gene rearrangement, evident also from still active internal translation start in <i>lmbN</i>, and indicating the direction of lincosamide biosynthesis evolution. The <i>in vitro</i> test with LmbN-CP, LmbC and the newly identified <i>S. lincolnensis</i> phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a <i>holo</i>-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.</p></div

Evidence of two translation starts in LmbN coding gene.

<p>(A) Western blot analysis of LmbN forms produced by <i>S. lincolnensis</i>, and heterologously in <i>S. coelicolor</i>. 1 and 7: standards of MW (His₈-tagged LmbN, His₈-tagged LmbN-ID, His₈-tagged LmbN-CP); 2: <i>S. lincolnensis</i> Δ<i>lmbN</i> mutant; 3: <i>S. lincolnensis</i> WT; 4: <i>S. coelicolor</i> M145 WT; 5: <i>S. coelicolor</i> M145 containing mutant form of <i>lmbN</i> with artificial stop codon introduced immediately downstream of the translation start 1; 6: <i>S. coelicolor</i> M145 containing native <i>lmbN gene</i>. (B) The detailed scheme of internal translation start (start 2) in <i>lmbN</i>. 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.</p

MS analysis of LmbN-CP.

<p>The mass corresponds to the mature LmbN-CP form. Percent values display abundance in comparison to the most abundant form of LmbN-CP in the sample. a.m.u.—atomic mass unit, N.o.—Not observed, N.A.—not applicable.</p><p>MS analysis of LmbN-CP.</p

Comparative analysis of lincomycin and celesticetin biosynthetic gene clusters.

<p>The genes coding for proteins with already confirmed/published functions are marked (C), the functions of other encoded proteins were assigned either according to sequence analysis combined with the prediction (P) in literature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118850#pone.0118850.ref014" target="_blank">14</a>], or based on BLAST analysis only (S). The genes in bold were selected for further testing. For shared homologous genes (below the bold line) the percentage of mutual identity of encoded proteins is shown, for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118850#pone.0118850.s001" target="_blank">S1 Document</a>. C8—octose; LIN—lincomycin; CEL—celesticetin;</p><p><i>*⁾</i>—the relevant function, biosynthetic step assignment and sequence identity value concern only the marked gene; MFS—major facilitator superfamily; salicylate—both biosynthesis and attachment of the salicylate unit; AS—biosynthesis of amino sugar; PPL—biosynthesis of PPL</p><p>Comparative analysis of lincomycin and celesticetin biosynthetic gene clusters.</p