Unifying and versatile features of flavin-dependent monooxygenases: Diverse catalysis by a common C4a-(hydro)peroxyflavin

Phintha, Aisaraphon; Chaiyen, Pimchai

doi:10.1016/j.jbc.2023.105413

Cited by 9 publications

(4 citation statements)

References 132 publications

(258 reference statements)

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“…The halogenase activity is particularly challenging because only a limited number of halogenases are available in the database. [14] We believe that this strategy and our mechanistic insights will provide a versatile biocatalytic halogenation platform because unlimited flavindependent monooxygenases with different protein foldings and substrate binding sites are available [44] for creating new halogenase scaffolds for a wide variety of substrate classes in the future.…”

Section: Discussionmentioning

confidence: 99%

On the Mechanisms of Hypohalous Acid Formation and Electrophilic Halogenation by Non‐Native Halogenases

Prakinee,

Lawan,

Phintha

et al. 2024

Angew Chem Int Ed

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Enzymatic electrophilic halogenation is a mild tool for functionalization of diverse organic compounds. Only a few groups of native halogenases are capable of catalyzing such a reaction. Here, we used a mechanism‐guided strategy to discover the electrophilic halogenation activity catalyzed by non‐native halogenases. As the ability to form a hypohalous acid (HOX) is key for halogenation, flavin‐dependent monooxygenases/oxidases capable of forming C4a‐hydroperoxyflavin (FlC4a‐OOH) such as dehalogenase, hydroxylases, luciferase and pyranose‐2‐oxidase (P2O), and flavin reductase capable of forming H2O2 were explored for their abilities to generate HOX in situ. Transient kinetic analyses using stopped‐flow spectrophotometry/ fluorometry and product analysis indicate that FlC4a‐OOH in dehalogenases, selected hydroxylases and luciferases, but not in P2O can form HOX; however, the HOX generated from FlC4a‐OOH cannot halogenate their substrates. Remarkably, in situ H2O2 generated by P2O can form HOI and also iodinate various compounds. Because not all enzymes capable of forming FlC4a‐OOH canreact with halides to form HOX, QM/MM calculations, site‐directed mutagenesis and structural analysis were carried out to elucidate the mechanism underlying the HOX formation and characterize the active site environment. Our findings shed light on identifying new halogenase scaffolds besides the currently known enzymes and have invoked a new mode of chemoenzymatic halogenations

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Section: Discussionmentioning

confidence: 99%

On the Mechanisms of Hypohalous Acid Formation and Electrophilic Halogenation by Non‐Native Halogenases

Prakinee,

Lawan,

Phintha

et al. 2024

Angew Chem Int Ed

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“…This in turn dictates that a concerted reaction between O 2 and carbon in organic compounds is spin-forbidden [ 3 ]. However, evolution has generated a significant number of enzymes able to oxygenate organic substrates by the prior activation of molecular oxygen to a 1-electron reduced form (O 2 − ), either by coopting an organic radical (e.g., a flavin nucleotide), or deploying a transition element (characterised by partially filled outer-bonding orbitals), which may (e.g., heme-coordinated iron) or may not (e.g., nonheme iron) be coordinated into an organic cofactor [ 4 , 5 ].…”

Section: Introductionmentioning

confidence: 99%

The Role of Dioxygen in Microbial Bio-Oxygenation: Challenging Biochemistry, Illustrated by a Short History of a Long Misunderstood Enzyme

Willetts

2024

Microorganisms

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A Special Issue of Microorganisms devoted to ‘Microbial Biocatalysis and Biodegradation’ would be incomplete without some form of acknowledgement of the many important roles that dioxygen-dependent enzymes (principally mono- and dioxygenases) play in relevant aspects of bio-oxygenation. This is reflected by the multiple strategic roles that dioxygen -dependent microbial enzymes play both in generating valuable synthons for chemoenzymatic synthesis and in facilitating reactions that help to drive the global geochemical carbon cycle. A useful insight into this can be gained by reviewing the evolution of the current status of 2,5-diketocamphane 1,2-monooxygenase (EC 1.14.14.108) from (+)-camphor-grown Pseudomonas putida ATCC 17453, the key enzyme that promotes the initial ring cleavage of this natural bicyclic terpene. Over the last sixty years, the perceived nature of this monooxygenase has transmogrified significantly. Commencing in the 1960s, extensive initial studies consistently reported that the enzyme was a monomeric true flavoprotein dependent on both FMNH2 and nonheme iron as bound cofactors. However, over the last decade, all those criteria have changed absolutely, and the enzyme is currently acknowledged to be a metal ion-independent homodimeric flavin-dependent two-component mono-oxygenase deploying FMNH2 as a cosubstrate. That transition is a paradigm of the ever evolving nature of scientific knowledge.

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“…The active protein complex LuxAB is a heterodimer of α (LuxA) and β (LuxB) subunits. As a flavin-dependent monooxygenase [13],…”

Section: Introductionmentioning

confidence: 99%

“…The active protein complex LuxAB is a heterodimer of α (LuxA) and β (LuxB) subunits. As a flavin-dependent monooxygenase [13], LuxAB binds reduced flavin mononucleotide (FMNH 2 ) and utilizes molecular oxygen to convert a long chain aldehyde to a fatty acid and emit light. Alongside the luxA and luxB genes, corresponding operons also contain the genes encoding a NADPH-dependent acyl protein reductase ( luxC ), an acyl-transferase ( luxD ), and an acyl-protein synthetase ( luxE ), usually in the order luxCDABE [7,8].…”

Section: Introductionmentioning

confidence: 99%

luxAgene fromEnhygromyxa salinaencodes a functional homodimeric luciferase

Yudenko

Bazhenov

Aleksenko

et al. 2023

Preprint

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Several clades of luminescent bacteria are known currently. They all contain similar lux operons, which include the genes luxA and luxB encoding a heterodimeric luciferase. The aldehyde oxygenation reaction is catalyzed by the subunit LuxA, while LuxB is inactive. Recently, genomic analysis identified a subset of bacterial species with rearranged lux operons lacking luxB. Here, we show that Escherichia coli transformed with a synthetic luxA gene from the reduced luxACDE operon of Enhygromyxa salina is luminescent upon addition of aldehydes. Overall, EsLuxA is less bright compared to luciferases from Aliivibrio fischeri (AfLuxAB) and Photorhabdus luminescens (PlLuxAB), and most active with medium-chain C4-C9 aldehydes. Crystal structure of EsLuxA determined at the resolution of 2.71 Å reveals a classical monooxygenase fold, and the protein preferentially forms a dimer in solution. The mobile loop residues 264-293, which form a β-hairpin or a coil in Vibrio harveyi LuxA, form α-helices in EsLuxA. Phylogenetic analysis shows EsLuxA and related proteins may be bacterial protoluciferases that arose prior to duplication of the luxA gene and its speciation to luxA and luxB in the previously described luminescent bacteria. Our work paves the way for discovery of new luciferases that have an advantage of being encoded by a single gene.

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Unifying and versatile features of flavin-dependent monooxygenases: Diverse catalysis by a common C4a-(hydro)peroxyflavin

Cited by 9 publications

References 132 publications

On the Mechanisms of Hypohalous Acid Formation and Electrophilic Halogenation by Non‐Native Halogenases

On the Mechanisms of Hypohalous Acid Formation and Electrophilic Halogenation by Non‐Native Halogenases

The Role of Dioxygen in Microbial Bio-Oxygenation: Challenging Biochemistry, Illustrated by a Short History of a Long Misunderstood Enzyme

luxAgene fromEnhygromyxa salinaencodes a functional homodimeric luciferase

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