Non‐Heme Dioxygenase Catalyzes Atypical Oxidations of 6,7‐Bicyclic Systems To Form the 6,6‐Quinolone Core of Viridicatin‐Type Fungal Alkaloids

Ishikawa, Noriyasu; Tanaka, Hidefumi; Koyama, Fumi; Noguchi, Hiroshi; Wang, Clay C. C.; Hotta, Kinya; Watanabe, Kenji

doi:10.1002/ange.201407920

Cited by 16 publications

(4 citation statements)

References 35 publications

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“…The dioxygenase AsqJ (ANID_09227) from A . nidulans has previously been shown to introduce a double bond in the same position of a phenylalanine moiety of an intermediate in the cyclopenin/4’-methoxycyclopenin biosynthetic pathway [ 26 ]. However, deletion of asqJ did not result in any changes of the final product of CcsA and CcsC.…”

Section: Resultsmentioning

confidence: 99%

Linker Flexibility Facilitates Module Exchange in Fungal Hybrid PKS-NRPS Engineering

et al. 2016

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Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) each give rise to a vast array of complex bioactive molecules with further complexity added by the existence of natural PKS-NRPS fusions. Rational genetic engineering for the production of natural product derivatives is desirable for the purpose of incorporating new functionalities into pre-existing molecules, or for optimization of known bioactivities. We sought to expand the range of natural product diversity by combining modules of PKS-NRPS hybrids from different hosts, hereby producing novel synthetic natural products. We succeeded in the construction of a functional cross-species chimeric PKS-NRPS expressed in Aspergillus nidulans. Module swapping of the two PKS-NRPS natural hybrids CcsA from Aspergillus clavatus involved in the biosynthesis of cytochalasin E and related Syn2 from rice plant pathogen Magnaporthe oryzae lead to production of novel hybrid products, demonstrating that the rational re-design of these fungal natural product enzymes is feasible. We also report the structure of four novel pseudo pre-cytochalasin intermediates, niduclavin and niduporthin along with the chimeric compounds niduchimaeralin A and B, all indicating that PKS-NRPS activity alone is insufficient for proper assembly of the cytochalasin core structure. Future success in the field of biocombinatorial synthesis of hybrid polyketide-nonribosomal peptides relies on the understanding of the fundamental mechanisms of inter-modular polyketide chain transfer. Therefore, we expressed several PKS-NRPS linker-modified variants. Intriguingly, the linker anatomy is less complex than expected, as these variants displayed great tolerance with regards to content and length, showing a hitherto unreported flexibility in PKS-NRPS hybrids, with great potential for synthetic biology-driven biocombinatorial chemistry.

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Section: Resultsmentioning

confidence: 99%

Linker Flexibility Facilitates Module Exchange in Fungal Hybrid PKS-NRPS Engineering

et al. 2016

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“…1) and related compounds are produced by species in cladistically different sections of Aspergillus. It is produced by Aspergillus sclerotiorum in section Circumdati (Visagie et al a, b, c), Aspergillus jensenii in section Versicolores (reported as Aspergillus nidulans by Ishikawa et al 2014) and by A. fumigatus in section Fumigati (Frisvad and Dyer, unpublished). Penicillins (Fig.…”

Section: Unique Extrolites In Subgenus Fumigatimentioning

confidence: 95%

Chemodiversity in the genus Aspergillus

Frisvad

Larsen

2015

Appl Microbiol Biotechnol

114

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Isolates of Aspergillus species are able to produce a large number of secondary metabolites. The profiles of biosynthetic families of secondary metabolites are species specific, whereas individual secondary metabolite families can occur in other species, even those phylogenetically and ecologically unrelated to Aspergillus. Furthermore, there is a high degree of chemo-consistency from isolate to isolate in a species even though certain metabolite gene clusters are silenced in some isolates. Genome sequencing projects have shown that the diversity of secondary metabolites is much larger in each species than previously thought. The potential of finding even further new bioactive drug candidates in Aspergillus is evident, despite the fact that many secondary metabolites have already been structure elucidated and chemotaxonomic studies have shown that many new secondary metabolites have yet to be characterized. The genus Aspergillus is cladistically holophyletic but phenotypically polythetic and very diverse and is associated to quite different sexual states. Following the one fungus one name system, the genus Aspergillus is restricted to a holophyletic clade that include the morphologically different genera Aspergillus, Dichotomomyces, Phialosimplex, Polypaecilum and Cristaspora. Secondary metabolites common between the subgenera and sections of Aspergillus are surprisingly few, but many metabolites are common to a majority of species within the sections. We call small molecule extrolites in the same biosynthetic family isoextrolites. However, it appears that secondary metabolites from one Aspergillus section have analogous metabolites in other sections (here also called heteroisoextrolites). In this review, we give a genus-wide overview of secondary metabolite production in Aspergillus species. Extrolites appear to have evolved because of ecological challenges rather than being inherited from ancestral species, at least when comparing the species in the different sections of Aspergillus. Within the Aspergillus sections, secondary metabolite pathways seem to inherit from ancestral species, but the profiles of these secondary metabolites are shaped by the biotic and abiotic environment. We hypothesize that many new and unique section-specific small molecule extrolites in each of the Aspergillus will be discovered.

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“…4 In the reactions of Fe/2OG monooxygenases from plants, fungi, and bacteria, different fates of the Fe(III)-OH/R• state (alternatives to step 4) lead to halogenation, desaturation, cyclization, ring-expansion and stereoinversion of aliphatic carbons. [14][15][16][17][18] With respect to biocatalysis and pharmaceutical synthesis, the abilities of certain Fe/2OG monooxygenases to install a halogen leaving group 14, [19][20] for chemoselective fragment couplings or an epoxide electrophile [21][22][23] for covalent capture of biomolecular targets are among the most potentially valuable. The mechanism of the latter reaction type is less thoroughly understood than those of either hydroxylation or halogenation, providing motivation for this study.…”

Section: Introductionmentioning

confidence: 99%

Optimized Substrate Positioning Enables Switches in C–H Cleavage Site and Reaction Outcome in the Hydroxylation-Epoxidation Sequence Catalyzed by Hyoscyamine 6β-Hydroxylase

Wenger,

Martinie,

Ushimaru

et al. 2024

Preprint

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Hyoscyamine 6β-hydroxylase (H6H) is an Fe(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase that catalyzes the last two steps in the biosynthesis of scopolamine, a prolifically administered anti-nausea drug. After its namesake first reaction, H6H couples the newly installed C6-bonded oxygen to C7 to form the epoxide of scopolamine. Oxoiron(IV) (ferryl) intermediates initiate both reactions by cleaving C–H bonds, but it remains unclear how the enzyme switches target site and promotes (C6)O–C7 coupling in preference to C7 hydroxylation in the second step. In one possible epoxidation mechanism, the C6 oxygen would – analogously to mechanisms proposed for the Fe/2OG halogenases and, in a parallel study, N-acetylnorloline synthase (LolO) – coordinate as alkoxide to the C7–H-cleaving ferryl intermediate to enable alkoxyl coupling to the ensuing C7 radical. Here we provide structural and kinetic evidence that H6H instead exploits the distinct spatial dependencies of competitive C–H-cleavage (C6 vs C7) and C–O-coupling (oxygen rebound vs cyclization) steps to promote the two-step sequence without substrate coordination or repositioning for the epoxidation step. Structural comparisons of ferryl-mimicking vanadyl complexes of wild-type H6H and a variant that preferentially 7-hydroxylates instead of epoxidizing 6β-hydroxyhyoscyamine suggest that only a modest (~ 10°) shift in the Fe–O–H(C7) approach angle is sufficient to change the outcome. The observation that, in wild-type H6H, ²H₂O solvent also increases the C7-hydroxylation:epoxidation ratio by ~ 8-fold implies that the latter outcome requires cleavage of the alcohol O-H bond, which, unlike in the LolO oxacyclization, is not accomplished in advance of C–H cleavage.

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Non‐Heme Dioxygenase Catalyzes Atypical Oxidations of 6,7‐Bicyclic Systems To Form the 6,6‐Quinolone Core of Viridicatin‐Type Fungal Alkaloids

Cited by 16 publications

References 35 publications

Linker Flexibility Facilitates Module Exchange in Fungal Hybrid PKS-NRPS Engineering

Linker Flexibility Facilitates Module Exchange in Fungal Hybrid PKS-NRPS Engineering

Chemodiversity in the genus Aspergillus

Optimized Substrate Positioning Enables Switches in C–H Cleavage Site and Reaction Outcome in the Hydroxylation-Epoxidation Sequence Catalyzed by Hyoscyamine 6β-Hydroxylase

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