The genome of the erythromycin-producing bacterium Saccharopolyspora erythraea contains many orphan secondary metabolite gene clusters including two (nrps3 and nrps5) predicted to govern biosynthesis of nonribosomal peptide-based siderophores. We report here the production by S. erythraea, even under iron-sufficient conditions, of a 2,5-diketopiperazine siderophore candidate we have named erythrochelin. Deletion of the nonribosomal peptide synthetase (NRPS) gene ercD within the nrps5 cluster abolished erythrochelin production. The tetrapeptide backbone of erythrochelin (alpha-N-acetyl-delta-N-acetyl-delta-N-hydroxyornithine-serine-delta-N-hydroxyornithine-delta-N-acetyl-delta-N-hydroxyornithine) suggests an orthodox colinear model for erythrochelin assembly. Curiously, the delta-N-acetyltransferase required for erythrochelin biosynthesis is encoded within a remote NRPS-cluster (nrps1) whose own NRPS contains an inactivating mutation. Disruption of the nrps1 gene mcd abolished erythrochelin biosynthesis, which could then be restored by addition of synthetic L-delta-N-acetyl-delta-N-hydroxyornithine, confirming an unprecedented example of functional crosstalk between nrps clusters.

Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.

The protein phosphatase inhibitor RK-682 is one of a number of potentially valuable tetronate polyketide natural products. Understanding how the tetronate ring is formed has been frustrated by the inaccessibility of the putative substrates. We report the heterologous expression of rk genes in Saccharopolyspora erythraea and reconstitution of the RK-682 pathway using recombinant enzymes, and show that RkD is the enzyme required for RK-682 formation from acyl carrier protein-bound substrates.

A novel mechanism is proposed for ring formation in the biosynthetic pathway to thiotetronate antibiotics thiolactomycin and Tü 3010.

[reaction: see text] Participating acyl groups located at C-2 in glucosyl and related donors generally promote formation of 1,2-trans-glycosides. Reactions of some glucuronic acid donors with TMSN(3)/SnCl(4) or ROH/SnCl(4) gave only the 1,2-cis-glycoside. The stereoselectivity is consistent with participation of the C-6 group. The methodology was used for the synthesis of a Kdn2en mimetic with the alpha-configuration.

Caught in the act: Intermediates in the biosynthesis of lasalocid A are captured in vivo by malonyl carba(dethia)‐N‐acetyl cysteamine probes. These species constitute novel snapshots of the timing of ether and aromatic ring formation, thus providing valuable insights for the reconstruction and the engineering of polyether biosynthetic pathways.

The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr42, His76, and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg120 located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity.

Numerous natural products of clinical value are biosynthesized by polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs), which are multienzymes comprising modules of catalytic domains. The key players in each module are carrier proteins, which serve as attachment points for the growing substrate chains. Thus, the details of carrier protein-based substrate delivery to each active site are central to understanding chain assembly in these systems. In the enterobactin NRPS, communication between a peptidyl carrier protein (PCP) and the adjacent thioesterase (TE) domain occurs through formation of a compact complex. Using NMR, we show that the corresponding interaction between a PKS acyl carrier protein (ACP) and its downstream TE is fundamentally different: chain transfer occurs in the absence of a protein-protein interface, with contact limited to the substrate acyl terminus.