Direct Evidence for Carbanions and Covalent N5-Flavin-Carbanion Adducts as Catalytic Intermediates in the Oxidation of Nitroethane by d-Amino Acid Oxidase

Porter, David; Voet, Judith G.; Bright, Harold J.

doi:10.1016/s0021-9258(19)43784-8

Cited by 159 publications

(70 citation statements)

References 27 publications

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“…The DAAO reaction is described by the two-step oxidative deamination of the d -amino group on the amino acid (Scheme A). To begin with, DAAO oxidizes the amino acid to an α-imimo acid through a hydride transfer from the C α to FAD . This is followed by a nucleophilic substitution by an activated water and the subsequent oxidation to form the ketone .…”

Section: Resultsmentioning

confidence: 99%

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MftD Catalyzes the Formation of a Biologically Active Redox Center in the Biosynthesis of the Ribosomally Synthesized and Post-translationally Modified Redox Cofactor Mycofactocin

Ayikpoe

Latham

2019

J. Am. Chem. Soc.

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Mycofactocin (MFT) is a putative ribosomally synthesized and post-translationally modified (RiPP) redox cofactor. The biosynthesis of MFT is encoded by the gene cluster mftABCDEF. While processing of the precursor peptide by MftB, MftC, and MftE has been shown to result in the formation of the small molecule 3-amino-5-[(p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP), no activity has been shown for the putative dehydrogenase MftD and the putative glycosyltransferase MftF. In addition, evidence demonstrating that MFT is a redox cofactor has only been limited to the requirement of mft genes for ethanol assimilation in M. smegmatis mc2155. Here, we demonstrate that MftD catalyzes the oxidative deamination of AHDP, forming an α-keto moiety on the resulting molecule which we call premycofactocin (PMFT). We characterize PMFT by 1D and 2D nuclear magnetic resonance spectroscopy techniques and by high-resolution mass spectrometry data to solve its structure. We further characterized PMFT by cyclic voltammetry and found its midpoint potential to be ~255 mV. Lastly, we demonstrate that PMFT is a biologically active redox cofactor that oxidizes NADH bound by M. smegmatis carveol dehydrogenase (MsCDH) and can be used by MsCDH in the oxidation of carveol. These data demonstrate for the first time that PMFT functions as a biologically active redox mediator and provides the most direct evidence to date that MFT is a RiPP-derived redox cofactor.

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Section: Resultsmentioning

confidence: 99%

“…To begin with, DAAO oxidizes the amino acid to an α-imimo acid through a hydride transfer from the C α to FAD. 37 This is followed by a nucleophilic substitution by an activated water and the subsequent oxidation to form the ketone. 33 We rationalized that MftD could carry out a similar reaction mechanism, which we describe in Scheme 1B.…”

Section: ■ Resultsmentioning

confidence: 99%

MftD Catalyzes the Formation of a Biologically Active Redox Center in the Biosynthesis of the Ribosomally Synthesized and Post-translationally Modified Redox Cofactor Mycofactocin

Ayikpoe

Latham

2019

J. Am. Chem. Soc.

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“…Thus, there must exist an excited-state process with the enzyme-bound FAD in choline oxidase that is faster than any internal conversions from higher excited states to the lowest excited state. − The maximal λ ex peak at 399 nm rules out an oxidized flavin being present in the excited state and suggests, instead, the presence of a flavin C4a adduct with the protein. Typically, flavin C4a adducts have maximal absorbance in the 380–400 nm region of the absorption spectrum, ,− as compared to the 300–355 nm region that is commonly associated with flavin N5 adducts. − An alternative flavin species that exhibits a single absorption peak at ∼400 nm is the protonated cationic oxidized flavin. , However, the p K a value for the equilibrium of cationic and neutral oxidized flavin is highly acidic, with a value of ∼0.1 in the ground state and ∼1.8 in the excited state, which is not consistent with the formation of the photoinduced species observed in this study at pH 10.0 (Figure D) and its lack of formation at pH 6.0 (Figure C). , In agreement with the excited state flavin of choline oxidase at high pH not being the protonated oxidized FAD, no fluorescence emission at ∼660 nm was observed . While other exceptions to the Kasha–Vavilov rule are known for flavins, ,, they all have fluorescence excitation spectra resembling either the overall UV–visible absorption spectra or their low-energy portions.…”

Section: Discussionmentioning

confidence: 99%

A Metastable Photoinduced Protein–Flavin Adduct in Choline Oxidase, an Enzyme Not Involved in Light-Dependent Processes

Smitherman

Gadda

2020

J. Phys. Chem. B

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Photoinduced formation of protein–flavin adducts is crucial in photoresponsive proteins containing light–oxygen–voltage (LOV) domains. LOV proteins typically share an N-terminal sensor domain with FMN and a C-terminal effector domain such as a kinase, a phosphodiesterase, or a DNA-binding protein. Light absorption by FMN results in a covalent flavin–cysteine adduct, which allosterically translates to the linked effector domain. Photoinduced protein–flavin adducts have not been reported in enzymes not involved in light-dependent processes. Here, we have used fluorescence, pH effects, and mutagenesis to follow up a serendipitous observation of an unusual fluorescence excitation spectrum in choline oxidase at alkaline pH (M. Ghanem and G. Gadda, unpublished observations). Physiologically, choline oxidase oxidizes choline to betaine through two FAD-associated reactions and is not a photoenzyme. The enzyme-bound flavin showed a progressive shift of the fluorescence excitation maximum (λex) from 468 to 399 nm with increasing pH values between pH 6.0 and 10.0, consistent with a metastable photoinduced protein–flavin adduct. In contrast, the maximal λem was independent of pH, with values of ∼526 nm. For comparison, fluorescence spectra of FAD in bulk solution had maximal values differing by ≤2 nm at different pH values, with λex at 453 nm and λem at 527 nm. The unusual behavior of the enzyme persisted in the mutated S101A enzyme variant but was eliminated in the H466Q variant, suggesting that the photoinduced species is likely a C4a-N-histidyl-FAD. The results provide evidence that metastable photoinduced formation of a flavin–protein adduct can occur in an enzyme that is not a photoreceptor or a photoenzyme.

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“…12 N 5 is also the site of covalent addition by substrate carbanions. 13 In parallel, the bridgehead C 4a position is the site of covalent connection with thiols 14 and also with radical oxygen species, such as superoxide equivalents to yield 4a-peroxides (Fig. 3).…”

Section: Canonical Features Of Flavoenzymesmentioning

confidence: 99%

Flavoenzymes: Versatile catalysts in biosynthetic pathways

2013

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Riboflavin-based coenzymes, tightly bound to enzymes catalyzing substrate oxidations and reductions, enable an enormous range of chemical transformations in biosynthetic pathways. Flavoenzymes catalyze substrate oxidations involving amine and alcohol oxidations and desaturations to olefins, the latter setting up Diels-Alder cyclizations in lovastatin and solanapyrone biosyntheses. Both C4a and N5 of the flavin coenzymes are sites for covalent adduct formation. For example, the reactivity of dihydroflavins with molecular oxygen leads to flavin-4a-OOH adducts which then carry out a diverse range of oxygen transfers, including Baeyer-Villiger type ring expansions, olefin epoxidations, halogenations via transient HOCl generation, and an oxidative Favorskii rerrangement during enterocin assembly.

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Direct Evidence for Carbanions and Covalent N5-Flavin-Carbanion Adducts as Catalytic Intermediates in the Oxidation of Nitroethane by d-Amino Acid Oxidase

Cited by 159 publications

References 27 publications

MftD Catalyzes the Formation of a Biologically Active Redox Center in the Biosynthesis of the Ribosomally Synthesized and Post-translationally Modified Redox Cofactor Mycofactocin

MftD Catalyzes the Formation of a Biologically Active Redox Center in the Biosynthesis of the Ribosomally Synthesized and Post-translationally Modified Redox Cofactor Mycofactocin

A Metastable Photoinduced Protein–Flavin Adduct in Choline Oxidase, an Enzyme Not Involved in Light-Dependent Processes

Flavoenzymes: Versatile catalysts in biosynthetic pathways

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