<sup>19</sup>F NMR studies on γ-butyrobetaine hydroxylase provide mechanistic insights and suggest a dual inhibition mode

Leśniak, Robert K.; Rydzik, Anna M.; Kamps, Jos J. A. G.; Kahn, Amjad; Claridge, Timothy D. W.; Schofield, Christopher J.

doi:10.1039/c9cc06466d

Cited by 4 publications

(8 citation statements)

References 32 publications

(63 reference statements)

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“…Alkylation of IPNS_S55C was confirmed by MS (Quattro Premier XE, Waters, USA); the observed mass for IPNS_S55C (37,768 Da) demonstrated an increase by 128 Da when alkylated to give IPNS* (37,896 Da, BTFA-labeled IPNS_S55C). Note that the predicted mass difference based on the calculated mass of CH 2 C(O)CF 3 (110 Da) was higher by 18 Da than observed, suggesting that the ketone exists mainly in its hydrated form, as observed for a different protein (34,40,41), and consistent with our crystallization analyses (Fig. 7).…”

Section: Expression and Purification Of Ipns_s55c And Labeling Of Ipn...supporting

confidence: 89%

X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis

et al. 2021

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Isopenicillin N synthase (IPNS) catalyzes the unique reaction of l--(-aminoadipoyl)-l-cysteinyl-d-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts. RESULTSTo focus tr-SFX analyses on events associated with O 2 reaction rather than ACV binding and/or IPN release, we co-crystallized the anaerobic IPNS:Fe(II):ACV complex under anaerobic conditions to produce a dense microcrystal slurry (~3 m × 3 m × 60 m, needle

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Section: Expression and Purification Of Ipns_s55c And Labeling Of Ipn...supporting

confidence: 89%

X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis

et al. 2021

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“…It is often convenient to think of enzymatic reactions as occurring in a linear fashion with one step completing before the next initiates. However, it is increasingly apparent that many biological processes instead occur by shifts in the equilibrium of the relevant complexes. ,− The current results suggest that a form of this dynamic equilibrium concept is at the heart of sMMO regulation. Nature forms and protects the key oxidative intermediate by controlling the concentrations, lifetimes, and irreversibility of reactions of specific protein–protein complexes.…”

Section: Discussionmentioning

confidence: 69%

“…BTFA was used to introduce fluorine into a cysteine residue based on established protocols. ,− A 100 μM sample of purified K15C-5FW-MMOB, 1 mM tris(2-carboxyethyl)phosphine (TCEP), and 400 μM BTFA were added to 100 mM MOPS buffer pH 7, to a final volume of 2.5 mL. The solution was allowed to stir for at least 1 h at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

Soluble Methane Monooxygenase Component Interactions Monitored by ¹⁹F NMR

et al. 2021

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Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme capable of catalyzing the fissure of the C−H bond of methane and the insertion of one atom of oxygen from O 2 to yield methanol. Efficient multiple-turnover catalysis occurs only in the presence of all three sMMO protein components: hydroxylase (MMOH), reductase (MMOR), and regulatory protein (MMOB). The complex series of sMMO protein component interactions that regulate the formation and decay of sMMO reaction cycle intermediates is not fully understood. Here, the two tryptophan residues in MMOB and the single tryptophan residue in MMOR are converted to 5-fluorotryptophan (5FW) by expression in defined media containing 5-fluoroindole. In addition, the mechanistically significant N-terminal region of MMOB is 19 F-labeled by reaction of the K15C variant with 3-bromo-1,1,1trifluoroacetone (BTFA). The 5FW and BTFA modifications cause minimal structural perturbation, allowing detailed studies of the interactions with sMMOH using 19 F NMR. Resonances from the 275 kDa complexes of sMMOH with 5FW-MMOB and BTFA-K15C-5FW-MMOB are readily detected at 5 μM labeled protein concentration. This approach shows directly that MMOR and MMOB competitively bind to sMMOH with similar K D values, independent of the oxidation state of the sMMOH diiron cluster. These findings suggest a new model for regulation in which the dynamic equilibration of MMOR and MMOB with sMMOH allows a transient formation of key reactive complexes that irreversibly pull the reaction cycle forward. The slow kinetics of exchange of the sMMOH:MMOB complex is proposed to prevent MMOR-mediated reductive quenching of the high-valent reaction cycle intermediate Q before it can react with methane.

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“…The high chemical shift stems from the conjugated carboxylic and hydroxamic acid system as already reported for the identical chemical substructure. 37 , 38 The splitting of the proton signals at position C9/C9′, C10/C10′, and C11/C11′ can be explained by the isomerism of the hydroxamic acid (see Supplementary Fig. S10 ) resulting in different chemical shifts.…”

Section: Resultsmentioning

confidence: 99%

Chryseochelins—structural characterization of novel citrate-based siderophores produced by plant protecting Chryseobacterium spp.

Rehm

Vollenweider

et al. 2023

Metallomics

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Bacteria secrete siderophores whose function is to acquire iron. In recent years, the siderophores of several Chryseobacterium species were shown to promote the health and growth of various plants such as tomato or rice. However, the chemical nature of Chryseobacterium siderophores remained unexplored despite great interest. In this work, we present the purification and structure elucidation by NMR and MS/MS of chryseochelin A, a novel citrate-based siderophore secreted by three Chryseobacterium strains involved in plant protection. It contains the unusual building blocks 3-hydroxycadaverine and fumaric acid. Furthermore, the unstable structural isomer chryseochelin B and its stable derivative containing fatty acid chains, named chryseochelin C, were identified by mass spectrometric methods. The latter two incorporate an unusual ester connectivity to the citrate moiety showing similarities to achromobactin from the plant pathogen Dickeya dadantii. Finally, we show that chryseochelin A acts in a concentration-dependent manner against the plant-pathogenic Ralstonia solanacearum strain by reducing its access to iron. Thus, our study provides valuable knowledge about the siderophores of Chryseobacterium strains, which have great potential in various applications.

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¹⁹F NMR studies on γ-butyrobetaine hydroxylase provide mechanistic insights and suggest a dual inhibition mode

Abstract: 19F and 1H NMR studies on fluorine labelled γ-butyrobetaine hydroxylase provide mechanistic insight into substrate and ligand binding, suggesting cooperativity between two monomers.

Cited by 4 publications

References 32 publications

X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis

X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis

Soluble Methane Monooxygenase Component Interactions Monitored by ¹⁹F NMR

Chryseochelins—structural characterization of novel citrate-based siderophores produced by plant protecting Chryseobacterium spp.

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19F NMR studies on γ-butyrobetaine hydroxylase provide mechanistic insights and suggest a dual inhibition mode

Abstract: 19F and 1H NMR studies on fluorine labelled γ-butyrobetaine hydroxylase provide mechanistic insight into substrate and ligand binding, suggesting cooperativity between two monomers.

Cited by 4 publications

References 32 publications

X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis

X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis

Soluble Methane Monooxygenase Component Interactions Monitored by 19F NMR

Chryseochelins—structural characterization of novel citrate-based siderophores produced by plant protecting Chryseobacterium spp.

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Product

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¹⁹F NMR studies on γ-butyrobetaine hydroxylase provide mechanistic insights and suggest a dual inhibition mode

Soluble Methane Monooxygenase Component Interactions Monitored by ¹⁹F NMR