Characterization of the Cereulide NRPS α-Hydroxy Acid Specifying Modules:  Activation of α-Keto Acids and Chiral Reduction on the Assembly Line

Abstract

Several nonribosomal peptide natural products are composites of alpha-hydroxy acid and alpha-amino acid monomers. Cereulide, the emetic toxin from the human pathogen Bacillus cereus, and valinomycin, from Streptomyces spp., are closely related macrocyclic K+ ionophores. The macrocyclic core of each natural product contains alternating peptide (six) and ester (six) bonds, and their cyclododecadepsipeptide structures consist of a tetradepsipeptide unit repeated three times. Here we overexpress the cereulide NRPS alpha-hydroxy acid specifying modules from CesA and CesB and demonstrate that each contains an alpha-keto acid activating adenylation domain and a chiral alpha-ketoacyl-S-carrier protein reductase (alpha-KR). The logic used by the cereulide NRPS is likely at work in the valinomycin NRPS and may be the general strategy used in bacterial NRPSs to form alpha-hydroxy acid containing natural products.

The identification of bacillaene, the product of the PksX megacomplex in Bacillus subtilis

“…The one characterized example is formed by the ketoreductase (KR)-catalyzed reduction of a thioester-linked ␣-keto-acid (14), but no such KR is found in the initiation module. The second NRPS module inserts glycine to complete the dipeptide starter unit, and the third NRPS module inserts alanine as part of an N-(␤-OH-propionyl)-Ala spacer between the hexaene and the triene.…”

A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both α- and β-ketone reduction during chain growth

“…2A) with a K m of 403 nM. In the depsipeptide ␣-ketoacid-activating A domains, a neutral residue replaces the aspartate that interacts with the positively charged substrate amine in ␣-amino acid-activating A domains (9). This mutation is also observed in the ␣-KIC-activating PksJ A 1 domain, wherein a valine (V240) replaces the conserved aspartate, further supporting the preference for ␣-KIC as a substrate.…”

“…Among the few characterized examples is the fungal secondary metabolite enniatin, in which the ␣-hydroxyacid is directly adenylated for incorporation into the natural product (8). Alternatively, in the cereulide depsipeptide assembly-line machinery, ␣-ketoacids are activated and reduced with chiral specificity by an A domain-embedded KR to the corresponding ␣-hydroxyacids while tethered to a T domain on the assembly line (9). No such module organization (A-KR-T) is found in the dihydrobacillaene assembly line.…”

Fungal cyclooligomerdepsipeptides: From classical biochemistry to combinatorial biosynthesis

“…90 Interestingly, no similarity is detected between the hydroxycarboxylic acid-activating A 1 domain signatures of the fungal CODSs and the 2-ketocarboxylic acid-activating A domain signatures derived from bacterial depsipeptide NRPSs. 22,57,108,109 4.2.2 Amino acid activating domains (A 2 ). The 10-amino acid specificity codes of the A 2 domains of CODSs show a higher degree of variation (Table 2).…”

“…98,108,109,133 Instead of utilizing preformed 2-hydroxycarboxylic acids as substrates, the respective bacterial NRPS A domains activate and load the corresponding 2-ketocarboxylic acids onto the multienzymes. Here, the ketocarboxylic acyl-thioesters undergo stereospecific reduction in cis by a ketoreductase (KR) domain, yielding the D-or L-hydroxycarboxylic acyl-thioesters ready for condensation.…”