Biochemical Characterization of the Two-Component Flavin-Dependent Monooxygenase Involved in Valanimycin Biosynthesis

Li, Hao; Forson, Benedicta; Eckshtain-Levi, Meital; Valentino, Hannah; Campo, Julia S. Martín del; Tanner, John J.; Sobrado, Pablo

doi:10.1021/acs.biochem.0c00679

Cited by 10 publications

(8 citation statements)

References 36 publications

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“…Multisubstrate enzyme mechanisms are difficult to study with conventional techniques. Elucidating such mechanisms requires the determination of the binding order of the substrates − and the determination of inhibition modes by the substrate. Therefore, the use of ITC seemed worthwhile to gain mechanistic insights and to elicit the robustness of our approach.…”

Section: Resultssupporting

confidence: 65%

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

et al. 2021

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To apply enzymes in technical processes, a detailed understanding of the molecular mechanisms is required. Kinetic and thermodynamic parameters of enzyme catalysis are crucial to plan, model, and implement biocatalytic processes more efficiently. While the kinetic parameters, K m and k cat , are often accessible by optical methods, the determination of thermodynamic parameters requires more sophisticated methods. Isothermal titration calorimetry (ITC) allows the label-free and highly sensitive analysis of kinetic and thermodynamic parameters of individual steps in the catalytic cycle of an enzyme reaction. However, since ITC is susceptible to interferences due to denaturation or agglomeration of the enzymes, the homogeneity of the enzyme sample must always be considered, and this can be accomplished by means of dynamic light scattering (DLS) analysis. We here report on the use of an ITC-dependent work flow to determine both the kinetic and the thermodynamic data for a cofactor-dependent enzyme. Using a standardized approach with the implementation of sample quality control by DLS, we obtain high-quality data suitable for the advanced modeling of the enzyme reaction mechanism. Specifically, we investigated stereoselective reactions catalyzed by the NADPH-dependent ketoreductase Gre2p under different reaction conditions. The results revealed that this enzyme operates with an ordered sequential mechanism and is affected by substrate or product inhibition depending on the reaction buffer. Data reproducibility is ensured by specifying standard operating procedures, using programmed workflows for data analysis, and storing all data in a F.A.I.R. (findable, accessible, interoperable, and reusable) repository (https://doi.org/10.15490/fairdomhub.1.investigation.464.1). Our work highlights the utility for combined binding and kinetic studies for such complex multisubstrate reactions.

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Section: Resultssupporting

confidence: 65%

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

et al. 2021

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“…The pathway is likely initiated by Ady2, a putative dehydrogenase that recruits fatty acids from primary metabolism to generate an aldehyde, which would be converted to an aliphatic amine by the pyridoxal phosphate (PLP)-dependent transaminase Ady4. The amine would be N-hydroxylated by the two-component flavindependent monooxygenase Ady3/Ady10, as in the valanimycin biosynthesis mediated by VlmH/VlmR [15][16][17]. The hydroxylamine would be conjugated to alanyl-tRNA to form an ester intermediate by the function of the tRNA-utilizing enzyme Ady7, which is homologous to VlmA.…”

Section: Biosynthetic Origin Of the Unique Methyl Ester In Azodyrecinmentioning

confidence: 99%

“…A distinct mechanism is employed in the biosynthesis of valanimycin, an aliphatic azoxy natural product. This involves the N-hydroxylation of isobutylamine, mediated by the flavin-dependent monooxygenase VlmH [15][16][17], and the following formation of O-(ʟ-seryl)-isobutylhydroxylamine by the tRNA-utilizing enzyme VlmA [18]. This intermediate is hypothesized to be transformed into the azoxy bond-containing intermediate via an intramolecular rearrangement accompanied by a concomitant oxidation [18].…”

Section: Introductionmentioning

confidence: 99%

New azodyrecins identified by a genome mining-directed reactivity-based screening

Choirunnisa¹,

Arima²,

Abe³

et al. 2022

Beilstein J. Org. Chem.

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Only a few azoxy natural products have been identified despite their intriguing biological activities. Azodyrecins D–G, four new analogs of aliphatic azoxides, were identified from two Streptomyces species by a reactivity-based screening that targets azoxy bonds. A biological activity evaluation demonstrated that the double bond in the alkyl side chain is important for the cytotoxicity of azodyrecins. An in vitro assay elucidated the tailoring step of azodyrecin biosynthesis, which is mediated by the S-adenosylmethionine (SAM)-dependent methyltransferase Ady1. This study paves the way for the targeted isolation of aliphatic azoxy natural products through a genome-mining approach and further investigations of their biosynthetic mechanisms.

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“…FMOs are commonly found in natural product biosynthetic pathways, where they play a major role in the addition of functional groups essential for bioactivity. , This family has been categorized into eight classes, A–H, according to structural and mechanistic characteristics . Our group has been studying several members of class B FMOs as these enzymes perform highly specific oxidations useful for biomedical and biotechnological applications. − …”

Section: Introductionmentioning

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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Biochemical Characterization of the Two-Component Flavin-Dependent Monooxygenase Involved in Valanimycin Biosynthesis

Cited by 10 publications

References 36 publications

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

New azodyrecins identified by a genome mining-directed reactivity-based screening

Characterization of a Nitro-Forming Enzyme Involved in Fosfazinomycin Biosynthesis

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