A biosynthetic aspartate N-hydroxylase performs successive oxidations by holding intermediates at a site away from the catalytic center

Rotilio, L.; Boverio, Alessandro; Nguyễn, Quốc Thái; Mannucci, Barbara; Fraaije, Marco W.; Mattevi, Andrea

doi:10.1016/j.jbc.2023.104904

Cited by 2 publications

(13 citation statements)

References 45 publications

(63 reference statements)

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“…Notably, unlike PHBH, steady-state kinetic analysis revealed that OxaD performs two oxidation reactions to convert roquefortine C into roquefortine L, in agreement with a previous report (Figures and S3). Previous studies of other multiple-oxidizing FMOs hypothesized that these enzymes might utilize a “release–hydroxylation–recapture” mechanism, wherein the intermediate is released from the active site but remains bound to a “capture” site until another round of reduction occurs . We propose that the “capture” site does not significantly differ from the original substrate binding site and that the conformational change required to hold onto N -hydroxy-roquefortine C would be small.…”

Section: Discussionsupporting

confidence: 74%

“… 6 Previous studies of other multiple-oxidizing FMOs hypothesized that these enzymes might utilize a “release–hydroxylation–recapture” mechanism, wherein the intermediate is released from the active site but remains bound to a “capture” site until another round of reduction occurs. 65 We propose that the “capture” site does not significantly differ from the original substrate binding site and that the conformational change required to hold onto N -hydroxy-roquefortine C would be small. Based on the previous proposal and our spectral data that suggested the presence of a stable complex between N -hydroxy-roquefortine C and OxaD, we propose that N -hydroxy-roquefortine C remains bound to the enzyme ( Figure 6 F).…”

Section: Discussionmentioning

confidence: 93%

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Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase

Johnson,

Li,

Valentino

et al. 2024

Biochemistry

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OxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a "cautious" flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme−substrate and enzyme− intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.

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Section: Discussionsupporting

confidence: 74%

Section: Discussionmentioning

confidence: 93%

Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase

Johnson,

Li,

Valentino

et al. 2024

Biochemistry

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“…A k cat of 4.28 � 0.07 s À 1 and a K M of 0.82 � 0.06 mM were measured with L-aspartate, which are similar to previously reported values for homologous enzymes (Figure 1 & Table 1). [13,17] We observed little change on the k cat value with D-aspartate but noted a ~23-fold increase in the K M value, suggesting that CreE preferentially binds Laspartate (Figure 1 & Table 1). A previous report showed that SV2À NMO was not active with D-aspartate by monitoring the rate of NADPH oxidation.…”

Section: Steady-state Kinetic Analysismentioning

confidence: 86%

“…[11,16] However, CreE is an unusual member of this subclass, because the enzyme catalyzes three sequential hydroxylation reactions of L-aspartate to yield nitrosuccinate, in contrast to the typical single oxidation reaction of NMOs (Scheme 1B). [8,11,13,17] Here we present the kinetic characterization of CreE. Steady-state kinetic studies demonstrated that CreE is stereospecific for L-aspartate and that the oxidation is highly coupled.…”

Section: Introductionmentioning

confidence: 94%

“…V2 L-aspartate N-monooxygenase (SV2À NMO) shows that these enzymes are members of class B of the FMO family. [13] The structures of class B FMOs feature two Rossmann nucleotide binding folds, of which one is responsible for FAD binding and the other binds NADPH/NADP + . [11] The kinetic mechanism can be divided into two parts: the reductive and oxidative-half reactions.…”

Section: Introductionmentioning

confidence: 99%

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Kinetic Characterization and Identification of Key Active Site Residues of the L‐Aspartate N‐Hydroxylase, CreE

Johnson,

Valentino,

Sobrado

2024

ChemBioChem

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CreE is a flavin‐dependent monooxygenase (FMO) that catalyzes three sequential nitrogen oxidation reactions of L‐aspartate to produce nitrosuccinate, contributing to the biosynthesis of the antimicrobial and antiproliferative nautral product, cremeomycin. This compound contains a highly reactive diazo functional group in which the reaction of CreE is essential to its formation. Nitro and diazo functional groups can serve as potent electrophiles, important in some challenging nucleophilic addition reactions. Formation of these reactive groups positions CreE as a promising candidate for biomedical and synthetic applications. Here, we present the catalytic mechanism of CreE and the identification of active site residues critical to binding L‐aspartate, aiding in future enzyme engineering efforts. Steady‐state analysis demonstrated that CreE is very specific for NADPH over NADH and performs a highly coupled reaction with L‐aspartate. Analysis of the rapid‐reaction kinetics showed that flavin reduction is very fast, along with the formation of the oxygenating species, the C4a‐hydroperoxyflavin. The slowest step observed was the dehydration of the flavin. Structural analysis and site‐directed mutagenesis implicated T65, R291, and R440 in the binding L‐aspartate. The data presented describes the catalytic mechanism and the active site architecture of this unique FMO.

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A biosynthetic aspartate N-hydroxylase performs successive oxidations by holding intermediates at a site away from the catalytic center

Cited by 2 publications

References 45 publications

Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase

Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase

Kinetic Characterization and Identification of Key Active Site Residues of the L‐Aspartate N‐Hydroxylase, CreE

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