Structures, mechanisms and applications of flavin-dependent halogenases

Phintha, Aisaraphon; Prakinee, Kridsadakorn; Chaiyen, Pimchai

doi:10.1016/bs.enz.2020.05.009

Cited by 26 publications

(36 citation statements)

References 46 publications

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“…Either HOX or N 6 -X-Lys could be the donor of X + to carbon nucleophiles. − So far, nearly all FDHs are associated with halogenation of aromatic substrates (Figure S1). , …”

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confidence: 75%

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AoiQ Catalyzes Geminal Dichlorination of 1,3-Diketone Natural Products

et al. 2021

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Enzymes that can perform halogenation of aliphatic carbons are of significant interest to the synthetic and biocatalysis communities. Here we describe the characterization of AoiQ, a single-component flavin-dependent halogenase (FDH) that catalyzes gem-dichlorination of 1,3-diketone substrates in the biosynthesis of dichlorodiaporthin. AoiQ represents the first biochemically reconstituted FDH that can halogenate an enolizable sp 3 -hybridized carbon atom.

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“…Either HOX or N 6 -X-Lys could be the donor of X + to carbon nucleophiles. − So far, nearly all FDHs are associated with halogenation of aromatic substrates (Figure S1). , …”

supporting

confidence: 75%

“…12−15 So far, nearly all FDHs are associated with halogenation of aromatic substrates (Figure S1). 16,17 In contrast, the roles of FDHs in catalyzing halogenation of aliphatic carbon nucleophiles are largely unexplored. Activated sp 3 carbons, such as the α-carbon of a 1,3-diketo moiety (pK a ∼9), would in theory serve as a suitable nucleophile.…”

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confidence: 99%

AoiQ Catalyzes Geminal Dichlorination of 1,3-Diketone Natural Products

et al. 2021

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“…[35] Aromatic halogenation is generally catalyzed by flavindependent halogenases (FDHs) that contain a characteristic WxWxIP motif that is absent in FMOs. [36] While none of the four enzymes contains such motif, ThmC shows moderate sequence identity (57 %) to PtmN, [37] which was reported to catalyze chlorination on C6 of the indole ring during penitrem biosynthesis. To test the function of ThmC, we coexpressed ThmC with ThmABDFGHIJK in A. nidulans, which led to formation of 3 (Figure S8, v).…”

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confidence: 99%

Biosynthesis of Terpenoid–Pyrrolobenzoxazine Hybrid Natural Product CJ‐12662

et al. 2022

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The fungal natural product CJ-12662 is a structurally complex terpene-amino acid hybrid, and is a potent anthelmintic compound. The biosynthetic pathway of CJ-12662 is elucidated based on metabolite analysis from heterologous expression. We demonstrate the terpene portion is derived from successive P450-catalyzed oxidations of amorpha-4,11-diene, while three flavin-dependent enzymes are involved in morphing the esterified tryptophan into a chlorinated pyrrolobenzoxazine, utilizing a cascaded [1,2]-Meisenheimer rearrangement.

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“…FDHs belong to the class of two-component flavin-dependent monooxygenases which require a reductase component to generate reduced FAD (FADH − ) for a halogenase (monooxygenase) component to catalyze halogenation ( 14 , 15 , 16 , 17 , 18 , 19 ). Several X-ray structures of FDHs have been solved ( 18 ) which showed that all FDHs have two separated binding sites.…”

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Dissecting the low catalytic capability of flavin-dependent halogenases

Phintha

Prakinee

Jaruwat

et al. 2021

Journal of Biological Chemistry

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Although flavin-dependent halogenases (FDHs) are attractive biocatalysts, their practical applications are limited because of their low catalytic efficiency. Here, we investigated the reaction mechanisms and structures of tryptophan 6-halogenase (Thal) from Streptomyces albogriseolususing stopped-flow, rapid-quench flow, QM/MM calculations, crystallography and detection of intermediate (hypohalous acid (HOX)) liberation. We found that the key flavin intermediate, C4a-hydroperoxyflavin (C4aOOH-FAD), formed by Thal and other FDHs(tryptophan 7-halogenase (PrnA) and tryptophan 5-halogenase (PyrH)), can react with I-, Br-, and Cl-but not F-, to form C4a-hydroxyflavin and HOX. Our experiments revealed that I-reacts with C4aOOH-FAD the fastest with the lowest energy barrier, and have shown for the first time that a significant amount of the HOX formed leaks out as free HOX. This leakage is probably a major cause of low product coupling ratios in all FDHs. Site-saturation mutagenesis of Lys79 showed that changing Lys79 to any other amino acid resulted in an inactive enzyme. However, the levels of liberated HOX of these variants are all similar, implying that Lys79 probably does not form a chloramine or bromamine intermediateas previously proposed. Computational calculations revealed that Lys79 has an abnormally lower pKa compared to other Lys residues, implying that the catalytic Lys may act as a proton donor in catalysis. Analysis of new X-ray structures of Thal also explains why pre-mixing of FDHs with FADH- generally results in abolishment of C4aOOH-FAD formation. These findings reveal the hidden factors restricting FDHs capability which should be useful for future development of FDHs applications.

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Structures, mechanisms and applications of flavin-dependent halogenases

Cited by 26 publications

References 46 publications

AoiQ Catalyzes Geminal Dichlorination of 1,3-Diketone Natural Products

AoiQ Catalyzes Geminal Dichlorination of 1,3-Diketone Natural Products

Biosynthesis of Terpenoid–Pyrrolobenzoxazine Hybrid Natural Product CJ‐12662

Dissecting the low catalytic capability of flavin-dependent halogenases

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