Mechanism-guided tunnel engineering to increase the efficiency of a flavin-dependent halogenase

Prakinee, Kridsadakorn; Phintha, Aisaraphon; Visitsatthawong, Surawit; Lawan, Narin; Sucharitakul, Jeerus; Kantiwiriyawanitch, Chadaporn; Damborský, Jiřı́; Chitnumsub, P.; Pée, Karl‐Heinz van; Chaiyen, Pimchai

doi:10.1038/s41929-022-00800-8

Cited by 69 publications

(62 citation statements)

References 58 publications

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“…Early studies established that FDH catalysis initially mirrors flavoprotein monooxygenase catalysis in that an enzyme‐bound, reduced flavin adenine dinucleotide (FADH 2 ) cofactor reacts with O 2 to generate a hydroperoxy flavin intermediate [6] . In FDHs, this intermediate reacts with bound halide, typically bromide or chloride, to generate HOX, which migrates through the enzyme to a substrate binding pocket [7–9] . Most evidence now suggests that hydrogen bonding by a key active site lysine residue activates HOX for electrophilic halogenation, and precise substrate binding leads to site‐selective halogenation by this species [5, 10, 11] …”

Section: Figurementioning

confidence: 99%

The Single‐Component Flavin Reductase/Flavin‐Dependent Halogenase AetF is a Versatile Catalyst for Selective Bromination and Iodination of Arenes and Olefins**

et al. 2022

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Flavin‐dependent halogenases (FDHs) natively catalyze selective halogenation of electron rich aromatic and enolate groups. Nearly all FDHs reported to date require a separate flavin reductase to supply them with FADH2, which complicates biocatalysis applications. In this study, we establish that the single component flavin reductase/flavin dependent halogenase AetF catalyzes halogenation of a diverse set of substrates using a commercially available glucose dehydrogenase to drive its halogenase activity. High site selectivity, activity on relatively unactivated substrates, and high enantioselectivity for atroposelective bromination and bromolactonization was demonstrated. Site‐selective iodination and enantioselective cycloiodoetherification was also possible using AetF. The substrate and reaction scope of AetF suggest that it has the potential to greatly improve the utility of biocatalytic halogenation.

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Section: Figurementioning

confidence: 99%

The Single‐Component Flavin Reductase/Flavin‐Dependent Halogenase AetF is a Versatile Catalyst for Selective Bromination and Iodination of Arenes and Olefins**

et al. 2022

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“…7 In addition, Prakinee et al reduced the halogenating intermediate leakage from the active sites of tryptophan 6halogenase through engineered V82 residue on the tunnel bottleneck, which connects the two active sites of flavindependent halogenases. 8 On the other hand, the entrance of tunnels is the first point of interaction with the substrates, which could deeply influence the enzyme activity and selectivity. Li et al performed saturation mutagenesis on residues (T354, M242, and Y365) at the tunnel entrance of monoamine oxidase MAO-N, leading to 0.67 U/mg activity (no activity for the wild type) and 93.4% stereoselectivity for substrate 1,2,3,4-tetrahydro-1-phenylisoquinoline.…”

Section: ■ Introductionmentioning

confidence: 99%

“…revealed that the CYP2E1 tunnel bottleneck residues (H107, A108, and H109) could affect the ligand affinity with the substrate (arachidonic acid) through electrostatic interactions . In addition, Prakinee et al reduced the halogenating intermediate leakage from the active sites of tryptophan 6-halogenase through engineered V82 residue on the tunnel bottleneck, which connects the two active sites of flavin-dependent halogenases . On the other hand, the entrance of tunnels is the first point of interaction with the substrates, which could deeply influence the enzyme activity and selectivity.…”

Section: Introductionmentioning

confidence: 99%

Flexibility Regulation of Loops Surrounding the Tunnel Entrance in Cytochrome P450 Enhanced Substrate Access Substantially

Meng

Nie

et al. 2022

ACS Catal.

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In recent years, the engineering of flexible loops to improve enzyme properties has gained attention in biocatalysis. Herein, we report a loop engineering strategy to improve the stability of the substrate access tunnels, which reveals the molecular mechanism between loops and tunnels. Based on the dynamic tunnel analysis of CYP116B3, five positions (A86, T91, M108, A109, T111) in loops B-B′ and B′-C potentially affecting tunnel frequent occurrence were selected and subjected to simultaneous saturation mutagenesis. The best variant 8G8 (A86T/T91L/M108N/A109M/T111A) for the dealkylation of 7-ethoxycoumarin and the hydroxylation of naphthalene was identified with considerably increased activity (134-fold and 9-fold) through screening. Molecular dynamics simulations showed that the reduced flexibility of loops B-B′ and B′-C was responsible for increasing the stability of the studied tunnel. The redesign of loops B-B′ and B′-C surrounding the tunnel entrance provides loop engineering with a powerful and likely general method to kick on/off the substrate/product transportation.

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“…[6] In FDHs, this intermediate reacts with bound halide, typically bromide or chloride, to generate HOX, which migrates through the enzyme to a substrate binding pocket. [7][8][9] Most evidence now suggests that hydrogen bonding by a key active site lysine residue activates HOX for electrophilic halogenation, and precise substrate binding leads to site-selective halogenation by this species. [5,10,11] Nearly all FDHs reported to date require a separate flavin reductase to supply FADH 2 , [6,12] and this enzyme is typically driven by a glucose/glucose dehydrogenase cofactor regeneration system for biocatalysis applications (Figure 1A).…”

mentioning

confidence: 99%

The Single‐Component Flavin Reductase/Flavin‐Dependent Halogenase AetF is a Versatile Catalyst for Selective Bromination and Iodination of Arenes and Olefins**

et al. 2022

View full text Add to dashboard Cite

Flavin-dependent halogenases (FDHs) natively catalyze selective halogenation of electron rich aromatic and enolate groups. Nearly all FDHs reported to date require a separate flavin reductase to supply them with FADH 2 , which complicates biocatalysis applications. In this study, we establish that the single component flavin reductase/flavin dependent halogenase AetF catalyzes halogenation of a diverse set of substrates using a commercially available glucose dehydrogenase to drive its halogenase activity. High site selectivity, activity on relatively unactivated substrates, and high enantioselectivity for atroposelective bromination and bromolactonization was demonstrated. Siteselective iodination and enantioselective cycloiodoetherification was also possible using AetF. The substrate and reaction scope of AetF suggest that it has the potential to greatly improve the utility of biocatalytic halogenation.Flavin-dependent halogenases (FDHs) natively catalyze site-selective halogenation of electron rich aromatic and enolate groups in a diverse range of halogenated natural products. [1][2][3] This unique capability has led to extensive efforts to understand FDH mechanism and the origins of their site selectivity. [4,5] Early studies established that FDH catalysis initially mirrors flavoprotein monooxygenase catalysis in that an enzyme-bound, reduced flavin adenine dinucleotide (FADH 2 ) cofactor reacts with O 2 to generate a hydroperoxy flavin intermediate. [6] In FDHs, this intermediate reacts with bound halide, typically bromide or chloride, to generate HOX, which migrates through the enzyme to a substrate binding pocket. [7][8][9] Most evidence now suggests that hydrogen bonding by a key active site lysine residue activates HOX for electrophilic halogenation, and precise substrate binding leads to site-selective halogenation by this species. [5,10,11] Nearly all FDHs reported to date require a separate flavin reductase to supply FADH 2 , [6,12] and this enzyme is typically driven by a glucose/glucose dehydrogenase cofactor regeneration system for biocatalysis applications (Figure 1A). [13] The need for a separate flavin reductase complicates biocatalysis since these enzymes are not widely available and are typically produced in-house, they add to the protein waste that must be removed during product isolation, and they can lead to undesired background reactions. [14] Previously, our group demonstrated that genetically fusing the flavin reductase RebF to the FDH RebH improved halogenation yields from whole-cell biocatalysis, suggesting that increased local concentration of FADH 2 can improve the efficiency of biocatalysis relative to the free enzymes (Figure 1A). [15] A recent family-wide sequence/

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Mechanism-guided tunnel engineering to increase the efficiency of a flavin-dependent halogenase

Cited by 69 publications

References 58 publications

The Single‐Component Flavin Reductase/Flavin‐Dependent Halogenase AetF is a Versatile Catalyst for Selective Bromination and Iodination of Arenes and Olefins**

The Single‐Component Flavin Reductase/Flavin‐Dependent Halogenase AetF is a Versatile Catalyst for Selective Bromination and Iodination of Arenes and Olefins**

Flexibility Regulation of Loops Surrounding the Tunnel Entrance in Cytochrome P450 Enhanced Substrate Access Substantially

The Single‐Component Flavin Reductase/Flavin‐Dependent Halogenase AetF is a Versatile Catalyst for Selective Bromination and Iodination of Arenes and Olefins**

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