Cytochrome P450 Catalyzes Benzene Ring Formation in the Biosynthesis of Trialkyl‐Substituted Aromatic Polyketides

Yu, Y. B.; Zhao, Er Juan; Wei, Wanqing; Xu, Zi Fei; Shi, Jing; Wu, Xuan; Zhang, Bo; Igarashi, Yasuhiro; Jiao, Rui Hua; Liang, Yong; Tan, Ren Xiang; Ge, Hui

doi:10.1002/ange.202214026

Cited by 2 publications

(4 citation statements)

References 39 publications

(60 reference statements)

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“…The proposal was supported by the recent report about the unusual cytochrome P450 monooxygenases LonP and GbnP, showing 46/91 and 46/97 identity%/coverage% to NftE 1 , which were veri ed to carry out the benzene ring formation in the biosynthesis of lorneic acids and gombapyrones [11]. Furthermore, the ΔnftF 1 mutant lost the capacity of producing compounds 3 and 4 and still kept the production of 1 and 2 (Fig.…”

Section: Identi Cation Of the Essential Genes From The Nft E 1 − I 1 ...mentioning

confidence: 54%

“…S7). However, no new product was generated from the assay consisting of 10 µM NftE 1 , 1 mM compound 5 or 6 or 7 or 8, 100 µg/mL ferredoxin (Fdx, spinach), 0.2 U/mL ferredoxin-NADP + reductase (FdR, spinach), 10 U/mL glucose dehydrogenase, 10 mM glucose, and 1 mM NADPH in 50 mM Tris.HCl Buffer (pH 7.4) at 30°C for 5 h. During our investigation, Yang et al con rmed that the cytochrome P450 LonP/Orf1-257 in lorneic acids biosynthesis catalyzes the biotransformation of an acyclic polyene substrate to the benzene ring [11]. As such, we re-investigated the activity of NftE 1 by using the alternative Fdx and FdR derived from cyanobacterium Synechococcus elongatus [24].…”

Section: Exploration Of the Nfte-mediate Polyketide Aromatic Cyclizationmentioning

confidence: 86%

“…1). Recently, the aromatic cores of lorneic acids and gombapyrones were veri ed to be formed by an unusual cytochrome P450 monooxygenase, which forms a benzene ring from a linear polyene chain o oaded from the type I PKS [11].…”

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: Identi Cation Of the Essential Genes From The Nft E 1 − I 1 ...mentioning

confidence: 54%

Section: Exploration Of the Nfte-mediate Polyketide Aromatic Cyclizationmentioning

confidence: 86%

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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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“…Although several P450s have the ability to catalyze two-step reactions for the biosynthesis of NPs, − this unusual two-step mechanism of BTG5 for constructing the unique bicyclo[3.2.2]nonane structure has never been reported. Therefore, this work advances the current knowledge of P450-catalyzed reactions. − …”

Section: Introductionmentioning

confidence: 99%

Discovery of a Fungal P450 with an Unusual Two-Step Mechanism for Constructing a Bicyclo[3.2.2]nonane Skeleton

Xu,

Yuan,

Yang

et al. 2024

J. Am. Chem. Soc.

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The successful biomimetic or chemoenzymatic synthesis of target natural products (NPs) and their derivatives relies on enzyme discovery. Herein, we discover a fungal P450 BTG5 that can catalyze the formation of a bicyclo[3.2.2]nonane structure through an unusual twostep mechanism of dimerization and cyclization in the biosynthesis of beticolin 1, whose bicyclo[3.2.2]nonane skeleton connects an anthraquinone moiety and a xanthone moiety. Further investigation reveals that BTG5-T318 not only determines the substrate selectivity but also alters the catalytic reactions, which allows the separation of the reaction to two individual steps, thereby understanding its catalytic mechanism. It reveals that the first heterodimerization undergoes the common oxidation process for P450s, while the second uncommon formal redox-neutral cyclization step is proved as a redox-mediated reaction, which has never been reported. Therefore, this work advances our understanding of P450-catalyzed reactions and paves the way for expansion of the diversity of this class of NPs through synthetic biology.

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Cytochrome P450 Catalyzes Benzene Ring Formation in the Biosynthesis of Trialkyl‐Substituted Aromatic Polyketides

Cited by 2 publications

References 39 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

Discovery of a Fungal P450 with an Unusual Two-Step Mechanism for Constructing a Bicyclo[3.2.2]nonane Skeleton

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