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1.
ACS Chem Biol ; 18(12): 2450-2456, 2023 Dec 15.
Article in English | MEDLINE | ID: mdl-37948749

ABSTRACT

Rieske oxygenases (ROs) from natural product biosynthetic pathways are a poorly studied group of enzymes with significant potential as oxidative functionalization biocatalysts. A study on the ROs JerL, JerP, and AmbP from the biosynthetic pathways of jerangolid A and ambruticin VS-3 is described. Their activity was successfully reconstituted using whole-cell bioconversion systems coexpressing the ROs and their respective natural flavin-dependent reductase (FDR) partners. Feeding authentic biosynthetic intermediates and synthetic surrogates to these strains confirmed the involvement of the ROs in hydroxymethylpyrone and dihydropyran formation and revealed crucial information about the RO's substrate specificity. The pronounced dependence of JerL and JerP on the presence of a methylenolether allowed the precise temporal assignment of RO catalysis to the ultimate steps of jerangolid biosynthesis. JerP and AmbP stand out among the biosynthetic ROs studied so far for their ability to catalyze clean tetrahydropyran desaturation without further functionalizing the formed electron-rich double bonds. This work highlights the remarkable ability of ROs to highly selectively oxidize complex molecular scaffolds.


Subject(s)
Oxygenases , Polyketides , Oxygenases/metabolism , Antifungal Agents , Reactive Oxygen Species , Catalysis , Oxidative Stress
2.
Org Biomol Chem ; 20(48): 9645-9649, 2022 12 14.
Article in English | MEDLINE | ID: mdl-36412217

ABSTRACT

Intramolecular oxa-Michael addition-catalysing cyclases are widespread in polyketide biosynthetic pathways. Although they have significant potential in biotechnology and chemoenzymatic synthesis of chiral heterocycles, they have only scarcely been studied. Here, we present detailed investigations on the selectivity profile of the pyran synthase PedPS7 showing that it combines broad substrate tolerance with high selectivity for the formation of up to two new stereocentres and relaxed precursor stereoisomer discrimination. Two of the four possible tetrahydropyran stereoisomers are reliably accessible by this enzyme. The results indicate fundamental differences between the individual subtypes of intramolecular oxa-Michael addition-catalysing cyclases.


Subject(s)
Polyketides , Pyrans , Pyrans/chemistry , Oxygen , Polyketides/chemistry , Catalysis , Stereoisomerism
3.
ACS Chem Biol ; 17(5): 1030-1037, 2022 05 20.
Article in English | MEDLINE | ID: mdl-35412301

ABSTRACT

Ketoreductases (KRs) are canonical domains of type I polyketide synthases (PKSs). They stereoselectively reduce ACP-bound ß-ketothioester intermediates and are responsible for a large part of the stereocenters in reduced polyketides. Albeit essential for the understanding and engineering of PKS, the specific effects of altering the polyketide part of KR precursors on their performance has rarely been studied. We present investigations on the substrate-dependent performance of six isolated KR domains using a library of structurally diverse surrogates for PKS thioester intermediates. A pronounced correlation between the polyketide structure and the KR performance was observed with activity and stereoselectivity diminishing with growing deviation from the natural KR precursor structure. The extent of this decrease and the profile of arising side products was characteristic for the individual KRs. Our results reinforce the importance of structure-KR performance relationships and suggest extended studies with isolated domains and whole PKS modules.


Subject(s)
Polyketide Synthases , Polyketides , Polyketide Synthases/metabolism , Polyketides/chemistry , Substrate Specificity
4.
RSC Adv ; 11(33): 20248-20251, 2021 Jun 03.
Article in English | MEDLINE | ID: mdl-35479892

ABSTRACT

The potential of polyketide synthase (PKS) domains for chemoenzymatic synthesis can often not be tapped due to their low stability and activity in vitro. In this proof-of-principle study, the immobilisation of the heterocycle-forming PKS domain AmbDH3 as a cross-linked enzyme aggregate (CLEA) is described. The AmbDH3-CLEA showed good activity recovery, stability and recyclability. Repetitive reactions on the semi-preparative scale were performed with high conversion and isolated yield. Similar to that observed for the free enzyme, the aggregate retained substrate tolerance and the ability for kinetic resolution. This first example of a successful enzymatic PKS domain immobilisation demonstrates that cross-linking can in principle be applied to this type of enzyme to increase its applicability for chemoenzymatic synthesis.

5.
Org Lett ; 22(13): 4955-4959, 2020 07 02.
Article in English | MEDLINE | ID: mdl-32610930

ABSTRACT

Studies on the biosynthetic processing of polyene thioester intermediates are complicated by limited access to appropriate substrate surrogates. We present a step-economic synthetic access to biomimetic ß-ketopolyene thioesters that is based on an Ir-catalyzed reductive Horner-Wadsworth-Emmons olefination. New ß-ketotriene and pentaenethioates of pantetheine and N-acetylcysteamine were exemplarily synthesized via short and concise routes. The usefulness of these compounds was demonstrated in an in vitro assay with the ketoreductase domain MycKRB from mycolactone biosynthesis.


Subject(s)
Alkenes/chemistry , Biomimetic Materials/chemistry , Biomimetic Materials/chemical synthesis , Esters/chemistry , Esters/chemical synthesis , Oxidoreductases/metabolism , Catalysis , Chemistry Techniques, Synthetic , Iridium/chemistry , Kinetics
6.
Nat Prod Rep ; 37(10): 1300-1315, 2020 10 01.
Article in English | MEDLINE | ID: mdl-32420573

ABSTRACT

Covering: 1977 to 2020The ambruticins and jerangolids are myxobacterial reduced polyketides, which are produced via highly unusual biosynthetic pathways containing a plethora of non-canonical enzymatic transformations. Since the discovery of the first congeners in the late 1970s, they have been in the focus of drug development due to their good antifungal activity and low toxicity in mammals, which result from interaction with an unusual innercellular target in fungi. Despite significant efforts, which have led to the development of various total syntheses, their structural complexity has yet avoided full exploitation of their pharmacological potential. This article summarises biological, total and semisynthetic as well as biosynthetic studies on both compounds. An outlook on the biosynthesis-based approaches to them and their derivatives is presented. Due to the structural and biosynthetic characteristics of the ambruticins and jerangolids, chemoenzymatic processes that make use of their biosynthetic pathway enzymes are particularly promising to gain efficient access to derivative libraries for structure activity relationship studies.


Subject(s)
Antifungal Agents/chemical synthesis , Pyrans/chemical synthesis , Antifungal Agents/pharmacology , Biosynthetic Pathways , Chemistry Techniques, Synthetic , Enzymes/chemistry , Enzymes/metabolism , Molecular Structure , Multigene Family , Mycobacterium/chemistry , Mycobacterium/metabolism , Pyrans/chemistry , Pyrans/metabolism , Pyrans/pharmacology
7.
J Org Chem ; 83(22): 14091-14101, 2018 11 16.
Article in English | MEDLINE | ID: mdl-30284827

ABSTRACT

A highly convergent access to the late-stage biosynthetic intermediates projerangolid and jerangolid E is presented, and its utility is demonstrated by the synthesis of novel non-natural jerangolid derivatives. The key steps are fragment couplings by Julia-Kocienski olefination and olefin cross metathesis, as well as a stereoselective tetrahydropyran formation by intramolecular oxa-Michael addition. Bioconversion experiments with the tailoring O-methyltransferase JerF confirmed its proposed biosynthetic role and revealed relaxed substrate specificity of this enzyme as well as tolerance to organic cosolvents.


Subject(s)
Deltaproteobacteria/metabolism , Polyketides/chemistry , Polyketides/metabolism , Deltaproteobacteria/chemistry , Molecular Structure
8.
Chembiochem ; 19(10): 1006-1011, 2018 05 18.
Article in English | MEDLINE | ID: mdl-29517170

ABSTRACT

The divinylcyclopropane (DVC) fragment of the ambruticins is proposed to be formed by a unique polyene cyclisation mechanism, in which the unusual didomain AmbG plays a key role. It is proposed to activate the branched thioester carboxylic acid resulting from polyene cyclisation and to transfer it to its associated acyl carrier protein (ACP). After oxidative decarboxylation, the intermediate is channelled back into polyketide synthase (PKS) processing. AmbG was previously annotated as an adenylation-thiolation didomain with a very unusual substrate selectivity code but has not yet been biochemically studied. On the basis of sequence and homology model analysis, we reannotate AmbG as a fatty acyl:adenylate ligase (FAAL)-acyl carrier protein didomain with unusual substrate specificity. The expected adenylate-forming activity on fatty acids was confirmed by in vitro studies. AmbG also adenylates a number of structurally diverse carboxylic acids, including functionalised fatty acids and unsaturated and aromatic carboxylic acids. HPLC-MS analysis and competition experiments show that AmbG preferentially acylates its ACP with long-chain hydrophobic acids and tolerates a π system and a branch near the carboxylic acid. AmbG is the first characterised example of a FAAL-ACP didomain that is centrally located in a PKS and apparently activates a polyketidic intermediate. This is an important step towards deeper biosynthetic studies such as partial reconstitution of the ambruticin pathway to elucidate DVC formation.


Subject(s)
Acyl Carrier Protein/metabolism , Bacteria/metabolism , Bacterial Proteins/metabolism , Biosynthetic Pathways , Ligases/metabolism , Polyketide Synthases/metabolism , Acyl Carrier Protein/chemistry , Bacteria/chemistry , Bacterial Proteins/chemistry , Cyclopropanes/metabolism , Decarboxylation , Ligases/chemistry , Oxidation-Reduction , Polyketide Synthases/chemistry , Protein Domains , Pyrans/metabolism , Substrate Specificity
9.
Angew Chem Int Ed Engl ; 57(1): 343-347, 2018 01 02.
Article in English | MEDLINE | ID: mdl-29084363

ABSTRACT

Oxygen-containing heterocycles are a common structural motif in polyketide natural products and contribute significantly to their biological activity. Here, we report structural and mechanistic investigations on AmbDH3, a polyketide synthase domain with dual activity as dehydratase (DH) and pyran-forming cyclase in ambruticin biosynthesis. AmbDH3 is similar to monofunctional DH domains, using H51 and D215 for dehydration. V173 was confirmed as a diagnostic residue for cyclization activity by a mutational study and enzymatic in vitro experiments. Similar motifs were observed in the seemingly monofunctional AmbDH2, which also shows an unexpected cyclase activity. Our results pave the way for mining of hidden cyclases in biosynthetic pathways. They also open interesting prospects for the generation of novel biocatalysts for chemoenzymatic synthesis and pyran-polyketides by combinatorial biosynthesis.

10.
Molecules ; 21(11)2016 Oct 29.
Article in English | MEDLINE | ID: mdl-27801873

ABSTRACT

We describe the characterisation of the O-methyltransferase JerF from the late stages of jerangolid biosynthesis. JerF is the first known example of an enzyme that catalyses the formation of a non-aromatic, cyclic methylenolether. The enzyme was overexpressed in E. coli and the cell-free extracts were used in bioconversion experiments. Chemical synthesis gave access to a series of substrate surrogates that covered a broad structural space. Enzymatic assays revealed a broad substrate tolerance and high regioselectivity of JerF, which makes it an attractive candidate for an application in chemoenzymatic synthesis with particular usefulness for late stage application on 4-methoxy-5,6-dihydro-2H-pyran-2-one-containing natural products.


Subject(s)
Methyltransferases/metabolism , Pyrans/metabolism , Biocatalysis , Escherichia coli/genetics , Methyltransferases/genetics , Stereoisomerism , Substrate Specificity
11.
Angew Chem Int Ed Engl ; 55(43): 13589-13592, 2016 10 17.
Article in English | MEDLINE | ID: mdl-27670141

ABSTRACT

The olefin shift is an important modification during polyketide biosynthesis. Particularly for type I cis-AT PKS, little information has been gained on the enzymatic mechanisms involved. We present our in vitro investigations on the olefin shift occurring during ambruticin biosynthesis. The unique, multifunctional domain AmbDH4 catalyzes consecutive dehydration, epimerization, and enoyl isomerization. The resulting 3-enethioate is removed from the equilibrium by α-methylation catalyzed by the highly specific C-methyltransferase AmbM. This thermodynamically unfavorable overall process is enabled by the high, concerted substrate specificity of the involved enzymes. AmbDH4 shows close relationship to DH domains and initial mechanistic studies suggest that the olefin shift occurs via a similar proton-shuttling mechanism as previously described for EI domains from trans-AT-PKS.


Subject(s)
Alkenes/metabolism , Hydro-Lyases/metabolism , Methyltransferases/metabolism , Alkenes/chemistry , Biocatalysis , Hydro-Lyases/chemistry , Methyltransferases/chemistry , Molecular Conformation , Pyrans/chemistry , Pyrans/metabolism
12.
Beilstein J Org Chem ; 12: 1512-50, 2016.
Article in English | MEDLINE | ID: mdl-27559404

ABSTRACT

This review highlights the biosynthesis of heterocycles in polyketide natural products with a focus on oxygen and nitrogen-containing heterocycles with ring sizes between 3 and 6 atoms. Heterocycles are abundant structural elements of natural products from all classes and they often contribute significantly to their biological activity. Progress in recent years has led to a much better understanding of their biosynthesis. In this context, plenty of novel enzymology has been discovered, suggesting that these pathways are an attractive target for future studies.

13.
Angew Chem Int Ed Engl ; 53(51): 14240-4, 2014 Dec 15.
Article in English | MEDLINE | ID: mdl-25327645

ABSTRACT

Hydropyran rings are a common structural motif in reduced polyketides. Information on their biosynthetic formation and particularly the biochemical characterization of the responsible enzymes has only been reported in few cases. The dehydratase domain AmbDH3 from the ambruticin polyketide synthase was investigated. Through in vitro assay of the recombinant domain with synthetically-derived substrate surrogates, it was shown that it has a second catalytic activity as a cyclase that performs oxa-conjugate addition. Probing AmbDH3 with synthetic substrate analogues revealed stereoselectivity and substrate tolerance in both substeps. This is the first characterization of a pyran-forming cyclase from a cis-AT PKS system and the first report of a polyketide synthase domain with this kind of dual activity. Finally, it was revealed that this domain shows potential for application in chemoenzymatic synthesis.


Subject(s)
Hydro-Lyases/metabolism , Enzyme Activation , Hydro-Lyases/chemistry , Hydro-Lyases/genetics , Molecular Conformation , Pyrans/chemistry , Pyrans/metabolism
14.
Beilstein J Org Chem ; 10: 634-640, 2014.
Article in English | MEDLINE | ID: mdl-24778714

ABSTRACT

Herein, we describe the syntheses of a complex biosynthesis-intermediate analogue of the potent antitumor polyketide borrelidin and of reference molecules to determine the stereoselectivity of the dehydratase of borrelidin polyketide synthase module 3. The target molecules were obtained from a common precursor aldehyde in the form of N-acetylcysteamine (SNAc) thioesters and methyl esters in 13 to 15 steps. Key steps for the assembly of the polyketide backbone of the dehydratase substrate analogue were a Yamamoto asymmetric carbocyclisation and a Sakurai allylation as well as an anti-selective aldol reaction. Reference compounds representing the E- and Z-configured double bond isomers as potential products of the dehydratase reaction were obtained from a common precursor aldehyde by Wittig olefination and Still-Gennari olefination. The final deprotection of TBS ethers and methyl esters was performed under mildly acidic conditions followed by pig liver esterase-mediated chemoselective hydrolysis. These conditions are compatible with the presence of a coenzyme A or a SNAc thioester, suggesting that they are generally applicable to the synthesis of complex polyketide-derived thioesters suited for biosynthesis studies.

16.
Beilstein J Org Chem ; 9: 56-63, 2013.
Article in English | MEDLINE | ID: mdl-23399592

ABSTRACT

Sugar moieties are present in a wide range of bioactive molecules. Thus, having versatile and fast methods for the decoration of biomimetic molecules with sugars is of fundamental importance. The glycosylation of peptoids and polyamines as examples of such biomimetic molecules is reported here. The method uses Cu-catalyzed azide alkyne cycloaddition to promote the reaction of azidosugars with either polyamines or peptoids. In addition, functionalized nucleic acids were attached to polyamines via the same route. Based on a modular solid-phase synthesis of peralkynylated peptoids with up to six alkyne groups, the latter were modified with azidosugar building blocks by using copper-catalyzed azide alkyne cycloadditions. In addition, the up-scaling of some particular azide-modified sugars is described.

19.
Nat Chem Biol ; 6(2): 99-101, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20081823

ABSTRACT

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 we show that RkD is the enzyme required for RK-682 formation from acyl carrier protein-bound substrates.


Subject(s)
Enzyme Inhibitors/metabolism , Phosphoprotein Phosphatases/antagonists & inhibitors , Saccharopolyspora/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Enzyme Inhibitors/chemistry , Molecular Structure , Multigene Family , Phosphoprotein Phosphatases/biosynthesis , Phosphoprotein Phosphatases/chemistry , Saccharopolyspora/chemistry , Saccharopolyspora/genetics , Substrate Specificity
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