Simplify, simplify, simplify! Pretubulysin (structure without the green substituents), a simplified tubulysin was prepared in the laboratory and also found in a natural myxobacterial source. This ...biosynthetic precursor of the tubulysins is not as active as tubulysins A and D but is still effective in picomolar concentrations against cancer cell lines.
Simplify, simplify, simplify! Pretubulysin (structure without the green substituents), a simplified tubulysin was prepared in the laboratory and also found in a natural myxobacterial source. This biosynthetic precursor of the tubulysins is not as active as tubulysins A and D but is still effective in picomolar concentrations against cancer cell lines.
Acquisition of new catalytic activity is a relatively rare evolutionary event. A striking example appears in the pathway to the antibiotic lankacidin, as a monoamine oxidase (MAO) family member, ...LkcE, catalyzes both an unusual amide oxidation, and a subsequent intramolecular Mannich reaction to form the polyketide macrocycle. We report evidence here for the molecular basis for this dual activity. The reaction sequence involves several essential active site residues and a conformational change likely comprising an interdomain hinge movement. These features, which have not previously been described in the MAO family, both depend on a unique dimerization mode relative to all structurally characterized members. Taken together, these data add weight to the idea that designing new multifunctional enzymes may require changes in both architecture and catalytic machinery. Encouragingly, however, our data also show LkcE to bind alternative substrates, supporting its potential utility as a general cyclization catalyst in synthetic biology.
Mutasynthesis couples the power of chemical synthesis with molecular biology to generate derivatives of medicinally valuable, natural products. Recently, this technique has been exploited by ...Cambridge-based biotech company Biotica Technology Ltd, and their collaborators, to generate promising new variants of the polyketide anti-cancer compounds rapamycin and borrelidin.
Modular polyketide synthases (PKSs) are multidomain multienzymes responsible for the biosynthesis in bacteria of a wide range of polyketide secondary metabolites of clinical value. The ...stereochemistry of these molecules is an attractive target for genetic engineering in attempts to produce analogues exhibiting novel therapeutic properties. The exchange of ketoreductase (KR) domains in model PKSs has been shown in several cases to predictably alter the configuration of the β‐hydroxy functionalities but not of the α‐methyl groups. By systematic screening of a broad panel of KR domains, we have identified two donor KRs that afford modification of α‐methyl group stereochemistry. To the best of our knowledge, this provides the first direct in vivo evidence of KR‐catalyzed epimerization. However, none of the introduced KRs afforded simultaneous alteration of methyl and hydroxy configurations in high yield. Therefore, swapping of whole modules might be necessary to achieve such changes in stereochemistry.
Manipulating polyketide stereochemistry: The stereochemistry of complex polyketides is intimately linked with bioactivity. Here, we identify ketoreductase domains in modular polyketide synthases that allow the rational modification of methyl‐group stereochemistry in a model system.
In the original version of this Article, the final concentration of riboflavin in the supplemented LB medium for recombinant LkcE expression was incorrectly stated as 1 g L
(this was the ...concentration of the stock solution) and should have read 10-50 mg L-
. This error has been corrected in both the PDF and HTML versions of the Article.
Polyketide natural products such as erythromycin and rapamycin are assembled on polyketide synthases (PKSs), which consist of modular sets of catalytic activities distributed across multiple protein ...subunits. Correct protein-protein interactions among the PKS subunits which are critical to the fidelity of biosynthesis are mediated in part by “docking domains” at the termini of the proteins. The NMR solution structure of a representative docking domain complex from the erythromycin PKS (DEBS) was recently solved, and on this basis it has been proposed that PKS docking is mediated by the formation of an intermolecular four-α-helix bundle. Herein, we report the genetic engineering of such a docking domain complex by replacement of specific helical segments and analysis of triketide synthesis by mutant PKSs in vivo. The results of these helix swaps are fully consistent with the model and highlight residues in the docking domains that may be targeted to alter the efficiency or specificity of subunit-subunit docking in hybrid PKSs.
A useful strategy: Deciphering biosynthesis by trans‐AT polyketide synthases (PKS) is challenging due to their highly nonlinear organization. Our analysis of the trans‐AT PKS responsible for ...sorangicin production in the myxobacterium Sorangium cellulosum So ce12 reinforces the utility of a combined phylogenetic/retrobiosynthetic approach for understanding this complex mode of biosynthesis.
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.
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