Microorganisms produce small molecules known as siderophores to scavenge iron from the environment. Insight into iron acquisition in myxobacteria has been provided recently by the sequencing of the ...gene cluster for the catecholate myxochelins A and B, from the myxobacterium Stigmatella aurantiaca Sg a15. The gene cluster contains enzymes (MxcCDEF) for assembly of 2,3-dihydroxybenzoic acid (DHBA), an amino transferase, MxcL, and a nonribosomal peptide synthetase (NRPS) subunit, MxcG. In the proposed pathway to the myxochelins, two molecules of DHBA are condensed with the two amino groups of lysine, which is itself tethered to the peptidyl carrier protein domain (PCP) of MxcG. The resulting thioester is then reduced by the NADPH-dependent reductase (Red) domain of MxcG to generate an aldehyde intermediate; subsequent Red-catalyzed reduction yields myxochelin A, while transamination by MxcL produces myxochelin B. Although myxochelin A has been obtained successfully in vitro, it has not been possible to date to reconstitute the transamination reaction to give myxochelin B nor to unequivocally establish the intermediacy of the aldehyde. We report here the successful biosynthesis of myxochelin B in vitro. Furthermore, we demonstrate for the first time the existence of an aldehyde intermediate in the four-electron reduction of a PCP-bound thioester. Finally, we show that the relative levels of myxochelin A and B are likely to be controlled by the direct competition of MxcL and the MxcG Red domain for a free aldehyde intermediate.
The ajudazols are antifungal secondary metabolites produced by a hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) multienzyme "assembly line" in the myxobacterium Chondromyces ...crocatus Cm c5. The most striking structural feature of these compounds is an isochromanone ring system; such an aromatic moiety is only known from two other complex polyketides, the electron transport inhibitor stigmatellin and the polyether lasalocid. The cyclization and aromatization reactions in the stigmatellin pathway are presumed to be catalyzed by a cyclase domain located at the end of the PKS, while the origin of the lasalocid benzenoid ring remains obscure. Notably, the ajudazol biosynthetic machinery does not incorporate a terminal cyclase, but instead a variant thioesterase (TE) domain. Here we present detailed phylogenetic and sequence analysis, coupled with experiments both in vitro and in vivo, that suggest that this TE promotes formation of the isochromanone ring, a novel reaction for this type of domain. As the ajudazol TE has homologues in several other secondary-metabolite pathways, these results are likely to be generalizable.
Spiroacetal substructures introduce important conformational constraints into bioactive polyketide natural products. New research reveals a two-enzyme team responsible for this molecular origami in ...reveromycin A biosynthesis. Highlights of this research are detailed.
Unusual biochemistry: The pathway to the antifungal ajudazols (ajudazol A is shown) has been deduced by cloning and sequencing of the gene cluster in the myxobacterium Chondromyces crocatus, coupled ...with insertional mutagenesis. Ajudazol biosynthesis incorporates several unusual features, including the first P450‐catalyzed dehydrogenation identified in bacterial metabolism.
Multienzyme docking in hybrid megasynthetases Richter, Carsten D; Broadhurst, R William; Weissman, Kira J ...
Nature chemical biology,
01/2008, Letnik:
4, Številka:
1
Journal Article
Recenzirano
Hybrid multienzyme systems composed of polyketide synthase (PKS) and nonribosomal polypeptide synthetase (NRPS) modules direct the biosynthesis of clinically valuable natural products in bacteria. ...The fidelity of this process depends on specific recognition between successive polypeptides in each assembly line-interactions that are mediated by terminal 'docking domains'. We have identified a new family of N-terminal docking domains, exemplified by TubCdd from the tubulysin system of Angiococcus disciformis An d48. TubCdd is homodimeric, which suggests that NRPS subunits in mixed systems self-associate to interact with partner PKS homodimers. The NMR structure of TubCdd reveals a new fold featuring an exposed beta-hairpin that serves as the binding site for the C-terminal docking domain of the partner polypeptide. The pattern of charged residues on the contact surface of the beta-hairpin is a key determinant of the interaction and seems to constitute a 'docking code' that can be used to alter binding affinity.
Fungi begin to reveal their secrets: Fungi elaborate a variety of polyketide secondary metabolites using three distinct biosynthetic machineries. Recent studies have started to reveal how these ...complicated multienzymes make specific biosynthetic “choices”.
The chondramides are mixed non-ribosomal peptide/polyketide secondary metabolites produced by the myxobacterium Chondromyces crocatus Cm c5, which exhibit strong cytotoxic activity. On the basis of ...their striking structural similarity to the marine depsipeptides jaspamides, the chondramides have been assumed to incorporate a (R)-β-tyrosine moiety, an expectation we confirm here. Thus, the recent sequencing of the chondramide biosynthetic gene cluster provided the opportunity to probe the shared origin of this unusual β-amino acid. We demonstrate here that (R)-β-tyrosine is produced directly from l-tyrosine by the aminomutase CmdF. Along with the tyrosine aminomutase SgcC4 from the C-1027 enediyne pathway, this enzyme belongs to a novel family of tyrosine aminomutases related to the ammonium lyase family of enzymes but exhibits opposite facial selectivity for the hydroxycinnamate intermediate. We also show that the adenylation (A) domain in the chondramide pathway, which activates the β-tyrosine building block, exhibits the required preference for (R)-β-tyrosine, further arguing against alternative origins for the moiety in the chondramides. Comparison to the (S)-β-tyrosine specific A domain SgcC1 should enhance our understanding of the structural and stereochemical determinants guiding amino acid selection by non-ribosomal peptide synthetase multienzymes.
Modular trans‐acyltransferase polyketide synthases (trans‐AT PKSs) are enzymatic assembly lines that biosynthesize complex polyketide natural products. Relative to their better studied cis‐AT ...counterparts, the trans‐AT PKSs introduce remarkable chemical diversity into their polyketide products. A notable example is the lobatamide A PKS, which incorporates a methylated oxime. Here we demonstrate biochemically that this functionality is installed on‐line by an unusual oxygenase‐containing bimodule. Furthermore, analysis of the oxygenase crystal structure coupled with site‐directed mutagenesis allows us to propose a model for catalysis, as well as identifying key protein‐protein interactions that support this chemistry. Overall, our work adds oxime‐forming machinery to the biomolecular toolbox available for trans‐AT PKS engineering, opening the way to introducing such masked aldehyde functionalities into diverse polyketides.
Benzolactone enamides, a number of which incorporate a methylated oxime moiety, are produced by a range of organisms, and constitute a family of cytotoxic natural products. Here, we determine how this capped oxime group is installed during assembly of the model polyketide lobatamide by a modular trans‐AT polyketide synthase and provide molecular insight into the responsible mono‐oxygenase domain by X‐ray crystallography.
Mycolactone, a polyketide toxin responsible for the extensive tissue destruction seen in Buruli ulcer, is assembled on a modular polyketide synthase (PKS). Despite operating on structurally different ...intermediates during synthesis, many of the ketoreductase (KR) domains of the mycolactone (MLS) PKS have identical sequences. This suggests that these enzymes might exhibit an unusually high level of substrate promiscuity. However, we show here that when recombinant mycolactone KR domains are tested with a range of surrogate substrates, their specificity closely matches that of KR domains derived from other PKS systems. In addition, our findings reinforce the role of substrate tethering for achieving stereochemical control in modular PKSs by affecting the delicate energetics of ketoreduction.
Crystal structure of a multienzyme: Ban et al. recently reported a breakthrough in understanding fatty acid synthesis in animals. Determination of a 3.2 Å crystal structure of porcine fatty acid ...synthase (FAS) reveals a multienzyme of startling architectural complexity.
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