Unexpected Role of a Short‐Chain Dehydrogenase/Reductase Family Protein in Type II Polyketide Biosynthesis

Gao, Yang; Zhao, Yuchun; Zhou, Jie; Yang, Maohua; Lin, Lin; Wang, Wenning; Tao, Meifeng; Deng, Zixin; Jiang, Ming

doi:10.1002/anie.202110445

Cited by 7 publications

(7 citation statements)

References 36 publications

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“…ActIV was characterized as a bifunctional cyclase-thioesterase via in vitro reconstitution of actinorhodin biosynthesis pathway, and it was also demonstrated with TE activity towards a model substrate of type II PKS, anthraquinone-2carboxylic acid-N-acetylcysteamine [14]. MrqD was suggested to function as a dedicated product releasing TE in murayaquinone biosynthesis based on the interpretations of gene deletion and sitedirected mutagenetic analysis [15]. With the information, we speculate that NftF 1 played the role of transacting TE to release the precursor chain of compound 3, and a subsequent hydroxylation on 3 provided 4.…”

Section: Exploration Of the Nfte-mediate Polyketide Aromatic Cyclizationmentioning

confidence: 99%

“…However, the MBL fold TEs were never reported in the type I PKSs that conventionally o oad chain by using an /ß hydrolase-type TE domain integrated at the C-terminus of PKS assembly line via thioester hydrolysis or O-C macrocyclization [13]. So far, only two cases of MBL fold hydrolases were experimentally investigated in the biosynthesis of type II polyketides actinorhodin and murayaquinone, in which ActIV and MrqD were characterized as MBL fold TEs for chain release based on genetic mutations and the in vitro analysis with an arti cial substrate [14,15]. Interestingly, these cognate genes appear popular in type II PKS BGCs, and they are usually annotated as ring cyclase/aromatase such as RslC2, AlnR and Ssfy2 derived from the biosynthesis of rishirilides, alnumycin and tetracycline SF2575 [16][17][18].…”

Section: Introductionmentioning

confidence: 99%

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Biosynthesis of trialkyl-substituted aromatic polyketide NFAT-133 involves unusual aromatization-mediating P450 monooxygenase and metallo-beta- lactamase fold thioesterase

Yang

Zhou

et al. 2023

Preprint

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Background The bacterial trialkyl-substituted aromatic polyketides are biosynthetically unique due to the unusual P450 monooxygenase-mediated aromatic core formation in the polyketide chains offloaded from type I polyketide synthase (PKS). As the representatives, TM-123 (1), veramycin A (2), NFAT-133 (3), and benwamycin I (4) were discovered from several Streptomyces species and they were characterized with antidiabetic and immunosuppressant activities. Though the biosynthesis of 1 − 3 were verified to be directed by the NFAT-133 biosynthetic gene cluster (nft BGC), it remains a mystery how 3 was generated during the biosynthesis. Results The biosynthesis of compounds 1 − 4 was verified to be directed by a cognate nft BGC in Streptomyces conglobatus. The PKS assembly logic of 1 − 4 was revised according to site-mutagenetic analysis of three PKS dehydratase domains. Gene deletion and mutant complementation of the five genes in nft BGC confirmed two essential genes of P450 monooxygenase nftE1 and metallo-beta-lactamase (MBL) fold hydrolase nftF1. Deletion of nftE1 led to abolishment of 1 − 4 and accumulation of four new products (5 − 8). Structural elucidation reveals 5 − 8 as the non-aromatic analogs of 1. Deletion of nftF1 gene resulted in disappearance of 3 and 4, while the production of 1 and 2 was not interrupted. Protein sequence analysis and structure-modeling by AlphaFold2 indicate that NftF1 is a MBL fold hydrolase. In vivo site-directed mutagenesis allowed identification of eight key residues in its active sites. Conclusion The PKS assembly logic of nft BGC is revised according to site-directed mutagenetic analysis. The benzene core of 1 − 4 should be formed through a NftE1-catalyzed oxidative-aromatization on the polyene chains offloaded from PKS. NftF1 should serve as a trans-acting thioesterase (TE) to mediate a premature chain-offloading from ACP7 to yield the nascent chain of compound 3. The key residues in the predicted active site of NftE1 were identified by in vivo site-directed mutations. Though the NftE1 homologs are widespread in type II PKSs, their functions are underexplored. As far as we know, NftE1 should represent the first MBL fold TE discovered from type I PKS.

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Section: Exploration Of the Nfte-mediate Polyketide Aromatic Cyclizationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Biosynthesis of trialkyl-substituted aromatic polyketide NFAT-133 involves unusual aromatization-mediating P450 monooxygenase and metallo-beta- lactamase fold thioesterase

Yang

Zhou

et al. 2023

Preprint

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“…Several studies have shown that CLF phylogeny more tightly correlates with the number of polyketide building block employed rather than the total carbon number. , During polyketide assembly the full-length reactive nascent poly-β-keto chain is often converted to the defined polycyclic skeleton as a result of a combination of ketoreductases, cyclases, and aromatases . Cyclases/aromatases function in a chaperone-like manner, catalyzing regioselective intramolecular aldol condensations en route to ring formation. , In the absence of cyclases/aromatases, spontaneous intramolecular cyclization of the highly reactive poly-β-keto intermediates can occur, leading to the formation of various shunt products. , …”

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confidence: 99%

Six Sets of Aromatic Polyketides Differing in Size and Shape Derive from a Single Biosynthetic Gene Cluster

Zhu

Zhang

et al. 2023

J. Nat. Prod.

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One new aromatic polyketide, prealnumycin B (1), and four known aromatic polyketides, K1115A (2), 1,6-dihydroxy-8-propylanthraquinone (DHPA, 3), phaeochromycin B (4), and (R)-7-acetyl-3,6-dihydroxy-8-propyl-3,4dihydronaphthalen-1(2H)-one (5), were isolated from the marine-derived Streptomyces sundarbansensis SCSIO NS01; these compounds represent four sets of aromatic polyketides differing in size and shape. A type II polyketide synthase (PKS) cluster, als, was identified by complete genome sequencing and was shown, by in vivo gene inactivation experiments in the wild-type (WT) NS01 strain and heterologous expression experiments, to encode the biosynthesis of compounds 1–5. Moreover, heterologous expression of the als cluster afforded three additional aromatic polyketides representing two different carbon skeletons, the new phaeochromycin L (6) and two known aromatic polyketides, phaeochromycins D (7) and E (8). These findings expand our knowledge of type II PKS machineries and their versatility in generating structurally diverse aromatic polyketides and highlight the power of type II PKSs in accessing new polyketides via ectopic expression in heterologous hosts.

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“…Specific to the production of aromatic polyketides, reminiscent here are the studies implicating the participation of NAD(P)(H)-dependent short chain dehydrogenases/reductases in catalyzing or facilitating polyketide cyclization/ aromatization reactions. 37,38 Complementary to in vivo gene manipulation experiments, the total in vitro reconstitution of large modular type I PKSs is now accessible which enables the discovery of new polyketide natural products and polyketide assembly line engineering. 39−41 Here, we employ the total in vitro reconstitution of the type I PKSs to discover a novel post-PKS tailoring enzymatic activity.…”

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confidence: 99%

“…The discovery of new tailoring enzymes that participate in the construction of polyketide antibiotics has traditionally relied on in vivo gene deletion experiments in native or heterologous hosts, the detection and isolation of biosynthetic intermediates, and assignment of the tailoring enzyme activities by gene complementation and model or native in vitro reactions. Specific to the production of aromatic polyketides, reminiscent here are the studies implicating the participation of NAD(P)(H)-dependent short chain dehydrogenases/reductases in catalyzing or facilitating polyketide cyclization/aromatization reactions. , Complementary to in vivo gene manipulation experiments, the total in vitro reconstitution of large modular type I PKSs is now accessible which enables the discovery of new polyketide natural products and polyketide assembly line engineering. − Here, we employ the total in vitro reconstitution of the type I PKSs to discover a novel post-PKS tailoring enzymatic activity. We demonstrate that the Plt PKSs themselves produce a biologically inactive alicyclic dihydrophloroglucinol polyketide product, a chemical class of products previously not reported to be produced by type I PKSs.…”

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confidence: 99%

A Nonfunctional Halogenase Masquerades as an Aromatizing Dehydratase in Biosynthesis of Pyrrolic Polyketides by Type I Polyketide Synthases

Niroula

Gutekunst

et al. 2022

ACS Chem. Biol.

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The bacterial modular type I polyketide synthases (PKSs) typically furnish nonaromatic lactone and lactam natural products. Here, by the complete in vitro enzymatic production of the polyketide antibiotic pyoluteorin, we describe the biosynthetic mechanism for the construction of an aromatic resorcylic ring by a type I PKS. We find that the pyoluteorin type I PKS does not produce an aromatic product, rather furnishing an alicyclic dihydrophloroglucinol that is later enzymatically dehydrated and aromatized. The aromatizing dehydratase is encoded in the pyoluteorin biosynthetic gene cluster (BGC), and its presence is conserved in other BGCs encoding production of pyrrolic polyketides. Sequence similarity and mutational analysis demonstrates that the overall structure and position of the active site for the aromatizing dehydratase is shared with flavin-dependent halogenases albeit with a loss in ability to perform redox catalysis. We demonstrate that the post-PKS dehydrative aromatization is critical for the antibiotic activity of pyoluteorin.

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Unexpected Role of a Short‐Chain Dehydrogenase/Reductase Family Protein in Type II Polyketide Biosynthesis

Cited by 7 publications

References 36 publications

Biosynthesis of trialkyl-substituted aromatic polyketide NFAT-133 involves unusual aromatization-mediating P450 monooxygenase and metallo-beta- lactamase fold thioesterase

Biosynthesis of trialkyl-substituted aromatic polyketide NFAT-133 involves unusual aromatization-mediating P450 monooxygenase and metallo-beta- lactamase fold thioesterase

Six Sets of Aromatic Polyketides Differing in Size and Shape Derive from a Single Biosynthetic Gene Cluster

A Nonfunctional Halogenase Masquerades as an Aromatizing Dehydratase in Biosynthesis of Pyrrolic Polyketides by Type I Polyketide Synthases

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