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

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

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

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 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

Identification of Fish Species and Targeted Genetic Modifications Based on DNA Analysis: State of the Art

Food adulteration is one of the most serious problems regarding food safety and quality worldwide. Besides misleading consumers, it poses a considerable health risk associated with the potential non-labeled allergen content. Fish and fish products are one of the most expensive and widely traded commodities, which predisposes them to being adulterated. Among all fraud types, replacing high-quality or rare fish with a less valuable species predominates. Because fish differ in their allergen content, specifically the main one, parvalbumin, their replacement can endanger consumers. This underlines the need for reliable, robust control systems for fish species identification. Various methods may be used for the aforementioned purpose. DNA-based methods are favored due to the characteristics of the target molecule, DNA, which is heat resistant, and the fact that through its sequencing, several other traits, including the recognition of genetic modifications, can be determined. Thus, they are considered to be powerful tools for identifying cases of food fraud. In this review, the major DNA-based methods applicable for fish meat and product authentication and their commercial applications are discussed, the possibilities of detecting genetic modifications in fish are evaluated, and future trends are highlighted, emphasizing the need for comprehensive and regularly updated online database resources

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