Unusual substrate and halide versatility of phenolic halogenase PltM

Mori, Shogo; Pang, A.H.; Chandrika, Nishad Thamban; Tsodikov, Oleg V.

doi:10.1038/s41467-019-09215-9

Cited by 43 publications

(52 citation statements)

References 39 publications

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“…This can be explained as in both structures the FAD-binding loop is part of a crystal contact, with the loops of two chains interacting with each other, generating a noncrystallographic twofold axis. In PltM, the conformation is very similar to that observed for 'closed' loop conformations (Mori et al, 2019). This is in contrast to the 'open' loop position in RebH (Bitto et al, 2008), where the loop is not in direct contact with the cofactor, leaving the FAD-binding site solvent exposed.…”

Section: Discussionsupporting

confidence: 51%

“…The first FDHs to be characterized were tryptophan halogenases (Trp halogenases; Keller et al, 2000;Yeh et al, 2005). In the following years, a variety of different halogenases were studied that can be grouped into indole halogenases (which include the Trp halogenases; Seibold et al, 2006;Heemstra & Walsh, 2008;Zeng & Zhan, 2011;Menon et al, 2016;Ma et al, 2017;Lingkon & Bellizzi, 2020;Domergue et al, 2019;Luhavaya et al, 2019), phenol halogenases (Buedenbender et al, 2009;Neumann et al, 2010;Zeng et al, 2013;Agarwal et al, 2014;Menon et al, 2017;Mori et al, 2019) and pyrrole halogenases (Hammer et al, 1997;Dorrestein et al, 2005;Yamanaka et al, 2012;Agarwal et al, 2014), depending on the classes of compounds that they halogenate.…”

Section: Introductionmentioning

confidence: 99%

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Structure of apo flavin-dependent halogenase Xcc4156 hints at a reason for cofactor-soaking difficulties

Widmann

Ismail

Sewald

et al. 2020

Acta Cryst Sect D Struct Biol

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Flavin-dependent halogenases regioselectively introduce halide substituents into electron-rich substrates under mild reaction conditions. For the enzyme Xcc4156 from Xanthomonas campestris, the structure of a complex with the cofactor flavin adenine dinucleotide (FAD) and a bromide ion would be of particular interest as this enzyme exclusively brominates model substrates in vitro. Apo Xcc4156 crystals diffracted to 1.6 Å resolution. The structure revealed an open substrate-binding site lacking the loop regions that close off the active site and contribute to substrate binding in tryptophan halogenases. Therefore, Xcc4156 might accept larger substrates, possibly even peptides. Soaking of apo Xcc4156 crystals with FAD led to crumbling of the intergrown crystals. Around half of the crystals soaked with FAD did not diffract, while in the others there was no electron density for FAD. The FAD-binding loop, which changes its conformation between the apo and the FAD-bound form in related enzymes, is involved in a crystal contact in the apo Xcc4156 crystals. The conformational change that is predicted to occur upon FAD binding would disrupt this crystal contact, providing a likely explanation for the destruction of the apo crystals in the presence of FAD. Soaking with only bromide did not result in bromide bound to the catalytic halide-binding site. Simultaneous soaking with FAD and bromide damaged the crystals more severely than soaking with only FAD. Together, these latter two observations suggest that FAD and bromide bind to Xcc4156 with positive cooperativity. Thus, apo Xcc4156 crystals provide functional insight into FAD and bromide binding, even though neither the cofactor nor the halide is visible in the structure.

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Section: Discussionsupporting

confidence: 51%

Section: Introductionmentioning

confidence: 99%

Structure of apo flavin-dependent halogenase Xcc4156 hints at a reason for cofactor-soaking difficulties

Widmann

Ismail

Sewald

et al. 2020

Acta Cryst Sect D Struct Biol

View full text Add to dashboard Cite

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“…However, certain anion preferences exist: The well described Trp-Fl-Hals RebH, Thal and PyrH prefer chlorination over bromination [24,25,27], while BrvH and VirX1 prefer bromination over chlorination [22,35]. For the three xcc-b100 enzymes, no chlorination was observed, and iodination was only found for Bmp5, PltM, and VirX1 [34,35,40,45] ( Table 1).…”

Section: Enzyme Diversity and Substrate Scopementioning

confidence: 99%

“…The authors found that PltM can halogenate several small phenolic and aniline derivatives. Encouraged by these findings, the study was successfully continued towards more bulky compounds including FDA approved drugs and natural products such as terbutaline, fenoterol, resveratrol, and catechin ( Figure 10) [45]. Notably, no halogenation activity was observed in the case of a nitrobenzene derivative due to the strongly electron withdrawing nitro group that prevented the halogenation of the substrate.…”

Section: Substrate Scope and Regioselectivitymentioning

confidence: 99%

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Recent Advances in Flavin-Dependent Halogenase Biocatalysis: Sourcing, Engineering, and Application

2019

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The introduction of a halogen atom into a small molecule can effectively modulate its properties, yielding bioactive substances of agrochemical and pharmaceutical interest. Consequently, the development of selective halogenation strategies is of high technological value. Besides chemical methodologies, enzymatic halogenations have received increased interest as they allow the selective installation of halogen atoms in molecular scaffolds of varying complexity under mild reaction conditions. Today, a comprehensive library of aromatic halogenases exists, and enzyme as well as reaction engineering approaches are being explored to broaden this enzyme family’s biocatalytic application range. In this review, we highlight recent developments in the sourcing, engineering, and application of flavin-dependent halogenases with a special focus on chemoenzymatic and coupled biosynthetic approaches.

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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 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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Unusual substrate and halide versatility of phenolic halogenase PltM

Cited by 43 publications

References 39 publications

Structure of apo flavin-dependent halogenase Xcc4156 hints at a reason for cofactor-soaking difficulties

Structure of apo flavin-dependent halogenase Xcc4156 hints at a reason for cofactor-soaking difficulties

Recent Advances in Flavin-Dependent Halogenase Biocatalysis: Sourcing, Engineering, and Application

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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