Flavin oxidation in flavin‐dependent <i>N</i>‐monooxygenases

Robinson, Reeder M.; Klancher, Catherine A.; Rodrı́guez, P.; Sobrado, Pablo

doi:10.1002/pro.3487

Cited by 10 publications

(15 citation statements)

References 38 publications

(74 reference statements)

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“…It has been previously shown that the pK a of the flavin N5 atom in SidA is modulated to stabilize the C4a-hydroperoxyflavin or to facilitate flavin oxidation. 24 We determined the pK a values for M101A in the absence of L-Orn, This interpretation is in agreement with the proposal of Setser et al that the purpose of the FAD conformational change from in to out is to eject NADP + in the last step of catalysis. 8 The M101A−NADP + complex perhaps represents a deadend state that is avoided by the wild-type enzyme but is made…”

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confidence: 90%

Structural Determinants of Flavin Dynamics in a Class B Monooxygenase

et al. 2020

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The ornithine hydroxylase known as SidA is a class B flavin monooxygenase that catalyzes the first step in the biosynthesis of hydroxamate-containing siderophores in Aspergillus f umigatus. Crystallographic studies of SidA revealed that the FAD undergoes dramatic conformational changes between out and in states during the catalytic cycle. We sought insight into the origins and purpose of flavin motion in class B monooxygenases by probing the function of Met101, a residue that contacts the pyrimidine ring of the in FAD. Steady-state kinetic measurements showed that the mutant variant M101A has a 25-fold lower turnover number. Pre-steady-state kinetic measurements, pH profiles, and solvent kinetic isotope effect measurements were used to isolate the microscopic step that is responsible for the reduced steady-state activity. The data are consistent with a bottleneck in the final step of the mechanism, which involves flavin dehydration and the release of hydroxy-Lornithine and NADP + . Crystal structures were determined for M101A in the resting state and complexed with NADP + . The resting enzyme structure is similar to that of wild-type SidA, consistent with M101A exhibiting normal kinetics for flavin reduction by NADPH and wild-type affinity for NADPH. In contrast, the structure of the M101A−NADP + complex unexpectedly shows the FAD adopting the out conformation and may represent a stalled conformation that is responsible for the slow kinetics. Altogether, our data support a previous proposal that one purpose of the FAD conformational change from in to out in class B flavin monooxygenases is to eject spent NADP + in preparation for a new catalytic cycle.

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confidence: 90%

Structural Determinants of Flavin Dynamics in a Class B Monooxygenase

et al. 2020

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“…DFT studies with SidA in complex with ornithine or lysine were consistent with homolytic cleavage of the Fl 4a -OOH and hydroxylation by hydroxyl radical transfer. The studies also suggested that ornithine binds with the N5-atom in the deprotonated form, which was later supported by pH studies [60]. The hydroxyl radical mechanism provides a logical explanation for the substrate selectivity of SidA.…”

Section: Hydroxylation Mechanismmentioning

confidence: 76%

New frontiers in flavin-dependent monooxygenases

Reis¹,

Li²,

Johnson³

et al. 2021

Archives of Biochemistry and Biophysics

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“…NMOs have been under investigation for many years with the best characterized examples being the l -ornithine monooxygenases SidA from Aspergillus fumigatus and PvdA from Pseudomonas aeruginosa. ,− ,− These enzymes perform a single hydroxylation of the N-atom on the side chain of l -ornithine forming hydroxylamine, which is part of the hydroxamate moiety of siderophores. Despite their prevalence in natural product pathways, little is known about NMOs outside of siderophore biosynthesis .…”

Section: Discussionmentioning

confidence: 99%

“…Thus, the amino acid sequences of FzmM, CreE (51% identity), SidA (15% identity), PvdA (14% identity), and KtzI (14% identity), a structural-characterized ornithine hydroxylase, were aligned to determine amino acid conservation (Figure ). Residues R121, Q80, and S610 of FzmM were aligned to R144, Q102, and S469 of SidA, which have been shown to be associated with FAD binding or C(4a)-hydroperoxyflavin stabilization. ,− The FAD-binding motif GXGXXG and some residues that are structurally associated with FAD binding such as P26 and W64 (P49 and W90 in SidA) are also present. , Key residues involved in NADPH binding in NMOs were absent in the FzmM amino acid sequence. This includes the SidA residues: R279 which is involved in NADPH selectivity, S257 essential for NADP + orientation, the Tyr-loop which undergoes major conformational changes as a part of NADPH binding, and the signature NMO “(F/V)ATGY” motif. ,,, The absence of these is significant as it suggests that dinucleotide substrate binding utilizes different structural features than that described in other NMOs.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Nitro-Forming Enzyme Involved in Fosfazinomycin Biosynthesis

Valentino

Sobrado

2021

Biochemistry

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N-hydroxylating monooxygenases (NMOs) are a subclass of flavindependent enzymes that hydroxylate nitrogen atoms. Recently, unique NMOs that perform multiple reactions on one substrate molecule have been identified. Fosfazinomycin M (FzmM) is one such NMO, forming nitrosuccinate from aspartate (Asp) in the fosfazinomycin biosynthetic pathway in some Streptomyces sp. This work details the biochemical and kinetic analysis of FzmM. Steady-state kinetic investigation shows that FzmM performs a coupled reaction with Asp (k cat , 3.0 ± 0.01 s −1 ) forming nitrosuccinate, which can be converted to fumarate and nitrite by the action of FzmL. FzmM displays a 70-fold higher k cat /K M value for NADPH compared to NADH and has a narrow optimal pH range (7.5−8.0). Contrary to other NMOs where the k red is rate-limiting, FzmM exhibits a very fast k red (50 ± 0.01 s −1 at 4 °C) with NADPH. NADPH binds at a K D value of ∼400 μM, and hydride transfer occurs with pro-R stereochemistry. Oxidation of FzmM in the absence of Asp exhibits a spectrum with a shoulder at ∼370 nm, consistent with the formation of a C(4a)hydroperoxyflavin intermediate, which decays into oxidized flavin and hydrogen peroxide at a rate 100-fold slower than the k cat . This reaction is enhanced in the presence of Asp with a slightly faster k ox than the k cat , suggesting that flavin dehydration or Asp oxidation is partially rate limiting. Multiple sequence analyses of FzmM to NMOs identified conserved residues involved in flavin binding but not for NADPH. Additional sequence analysis to related monooxygenases suggests that FzmM shares sequence motifs absent in other NMOs. 65 characterization of the nitro-forming NMO FzmM providing a 66 detailed report of its mechanism toward the formation of 67 nitrosuccinate.

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Flavin oxidation in flavin‐dependent N‐monooxygenases

Cited by 10 publications

References 38 publications

Structural Determinants of Flavin Dynamics in a Class B Monooxygenase

Structural Determinants of Flavin Dynamics in a Class B Monooxygenase

New frontiers in flavin-dependent monooxygenases

Characterization of a Nitro-Forming Enzyme Involved in Fosfazinomycin Biosynthesis

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