NMR studies of the non-haem Fe(II) and 2-oxoglutarate-dependent oxygenases

Mbenza, Naasson M.; Vadakkedath, Praveen G.; McGillivray, Duncan J.; Leung, Ivanhoe K. H.

doi:10.1016/j.jinorgbio.2017.08.032

Cited by 4 publications

(3 citation statements)

References 88 publications

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“…Having shown that the interaction between tPHD2 and CODD is preserved in the gas phase, we then carried out solution studies. NMR studies 21 , 22 using 15 N-labelled PHD2 181–402 showed that CODD and hyCODD bind with approximately equal affinity to apo -PHD2 181–402 and PHD2 181–402 ·Zn ( i.e. both saturated apo -PHD2 181–402 and PHD2 181–402 ·Zn at 2.7-fold excess) ( Fig.…”

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confidence: 98%

2-Oxoglutarate regulates binding of hydroxylated hypoxia-inducible factor to prolyl hydroxylase domain 2

et al. 2018

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2-Oxoglutarate regulates binding of hydroxylated hypoxia-inducible factor to prolyl hydroxylase domain 2

et al. 2018

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“…1 H nuclear magnetic resonance (NMR) spectroscopy is an established technique for the study of enzyme kinetics that has been used to characterise different enzyme systems including (but not limited to) carbohydrate-processing enzymes, 23 , 24 enzymes related to antibiotic resistance 25 and oxygenases. 26 , 27 1 H NMR spectroscopy enables the direct monitoring of reaction kinetics in real time and accurate, quantitative information can be obtained by following changes in the peak area of the resonances associated with the substrate and/or reaction product(s). In contrast, thermal shift assay is a simple and high-throughput method that can be used to study protein–ligand binding interactions by measuring the melting temperature of a protein by the use of a fluorescence dye that is sensitive to changes in hydrophobic environment.…”

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Development of NMR and thermal shift assays for the evaluation ofMycobacterium tuberculosisisocitrate lyase inhibitors

et al. 2017

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“…This establishes changes in protein dynamics essential for the sequentially ordered mechanism of FIH, raising the potential that dynamics in other αKG-dependent oxygenases may be functionally significant. 16,33 Decreased protein dynamics in FIH upon binding (M+αKG) reveal metal is required for binding-based inhibitor screens, as shown by a thermal stability assay of the binding of the inhibitor to FIH. The correlation between (M+αKG) binding and protein dynamics has important ramifications on the nature of substrate binding, catalysis, and the design and screening of selective inhibitors for FIH.…”

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Protein Flexibility of the α-Ketoglutarate-Dependent Oxygenase Factor-Inhibiting HIF-1: Implications for Substrate Binding, Catalysis, and Regulation

et al. 2019

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Protein dynamics are crucial for the mechanistically ordered enzymes to bind to their substrate in the correct sequence and perform catalysis. Factor-inhibiting HIF-1 (FIH) is a nonheme Fe(II) αketoglutarate-dependent oxygenase that is a key hypoxia (low p O 2) sensor in humans. As these hypoxia-sensing enzymes follow a multistep chemical mechanism consuming α-ketoglutarate, a protein substrate that is hydroxylated, and O 2 , understanding protein flexibility and the order of substrate binding may aid in the development of strategies for selective targeting. The primary substrate of FIH is the C-terminal transactivation domain (CTAD) of hypoxia-inducible factor 1α (HIF) that is hydroxylated on the side chain of Asn803. We assessed changes in protein flexibility connected to metal and αKG binding, finding that (M+αKG) binding significantly stabilized the cupin barrel core of FIH as evidenced by enhanced thermal stability and decreased protein dynamics as assessed by global amide hydrogen/deuterium exchange mass spectrometry and limited proteolysis. Confirming predictions of the consensus mechanism, (M+αKG) increased the affinity of FIH for CTAD as measured by titrations monitoring intrinsic tryptophan fluorescence.The decreased protein dynamics caused by (M+αKG) enforces a sequentially ordered substrate binding sequence in which αKG binds before CTAD, suggesting that selective inhibition may require inhibitors that target the binding sites of both αKG and the prime substrate. A consequence of the correlation between dynamics and αKG binding is that all relevant ligands must be included in binding-based inhibitor screens, as shown by testing permutations of M, αKG, and inhibitor.

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NMR studies of the non-haem Fe(II) and 2-oxoglutarate-dependent oxygenases

Cited by 4 publications

References 88 publications

2-Oxoglutarate regulates binding of hydroxylated hypoxia-inducible factor to prolyl hydroxylase domain 2

2-Oxoglutarate regulates binding of hydroxylated hypoxia-inducible factor to prolyl hydroxylase domain 2

Development of NMR and thermal shift assays for the evaluation ofMycobacterium tuberculosisisocitrate lyase inhibitors

Protein Flexibility of the α-Ketoglutarate-Dependent Oxygenase Factor-Inhibiting HIF-1: Implications for Substrate Binding, Catalysis, and Regulation

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