Gas tunnel engineering of prolyl hydroxylase reprograms hypoxia signaling in cells DOI Creative Commons
Peter Windsor,

Haiping Ouyang,

Joseph A. G. da Costa

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Aug. 7, 2023

Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O

Language: Английский

Sequential oxidation of L-lysine by a non-heme iron hydroxylase DOI Creative Commons

Edward S. Reynolds,

Thomas G. Smith, Anoop R. Damodaran

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 28, 2025

2-oxoglutarate-dependent non-heme iron hydroxylases offer a direct route to functionalizing C( sp 3 )–H bonds across diverse range of substrates, making them prime candidates for chemoenzymatic synthetic strategies. We demonstrate the ability L-lysine dioxygenase perform sequential oxidation and computationally explore structural elements that promote this reactivity.

Language: Английский

Citations

0
Gas tunnel engineering of prolyl hydroxylase reprograms hypoxia signaling in cells DOI Creative Commons
Peter Windsor,

Haiping Ouyang,

Joseph A. G. da Costa

et al.

Angewandte Chemie International Edition, Journal Year: 2024, Volume and Issue: 63(48)

Published: Aug. 22, 2024

Abstract Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O 2 ) concentrations. Here, we reprogrammed cellular hypoxia (low O signaling via gas tunnel engineering of prolyl hydroxylase (PHD2), a non‐heme iron dependent sensor. Using computational modeling protein techniques, identify critical residues therein that limit the flow PHD2’s catalytic core. We show systematic modification these can open constriction topology tunnel. kinetic stopped‐flow measurements with NO as surrogate diatomic gas, demonstrate up 3.5‐fold enhancement its association rate center tunnel‐engineered mutants. Our most effectively designed mutant displays 9‐fold enhanced efficiency ( k cat / K M =830±40 −1 s hydroxylating peptide mimic inducible transcription factor HIF‐1α, compared WT PHD2 =90±9 ). Furthermore, transfection plasmids express mutants HEK‐293T mammalian cells reveal significant reduction HIF‐1α downstream response transcripts under hypoxic conditions 1 % . Overall, studies highlight activation new pathway reprogram responses HIF cells.

Language: Английский

Citations

3
Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase DOI
Elizabeth R. Smithwick, Ambika Bhagi‐Damodaran, Anoop R. Damodaran

et al.

Methods in enzymology on CD-ROM/Methods in enzymology, Journal Year: 2024, Volume and Issue: unknown, P. 51 - 63

Published: Jan. 1, 2024

Language: Английский

Citations

2
Controllable Multihalogenation of a Non-native Substrate by the SyrB2 Iron Halogenase DOI

R. Hunter Wilson,

Sourav Chatterjee, Elizabeth R. Smithwick

et al.

ACS Catalysis, Journal Year: 2024, Volume and Issue: 14(17), P. 13209 - 13218

Published: Aug. 19, 2024

Geminal, multihalogenated functional groups are widespread in natural products and pharmaceuticals, yet no synthetic methodologies exist that enable selective multihalogenation of unactivated C–H bonds. Biocatalysts powerful tools for late-stage functionalization as they operate with high degrees regio-, chemo-, stereoselectivity. 2-Oxoglutarate (2OG)-dependent nonheme iron halogenases chlorinate brominate aliphatic bonds offer a solution achieving these challenging transformations. Here, we describe the ability halogenase, SyrB2, to controllably halogenate non-native substrate α-aminobutyric acid (Aba) yield monochlorinated, dichlorinated, trichlorinated products. These chemoselective outcomes achieved by controlling loading 2OG cofactor SyrB2 biocatalyst. Furthermore, using ferredoxin-based biological reductant electron transfer catalytic center demonstrate order-of-magnitude enhancement were previously inaccessible any single halogenase enzyme. We also apply strategies broaden SyrB2's reactivity scope include multibromination chemoenzymatic conversion ethyl side chain Aba an ethynyl group. show how steric hindrance induced successive addition halogen atoms on Aba's C4 carbon dictates degree hampering C3–C4 bond rotation within pocket. Overall, our work showcases potential facilitate multi–C–H chemistry.

Language: Английский

Citations

2
Gas tunnel engineering of prolyl hydroxylase reprograms hypoxia signaling in cells DOI Creative Commons
Peter Windsor,

Haiping Ouyang,

Joseph A. G. da Costa

et al.

Angewandte Chemie, Journal Year: 2024, Volume and Issue: 136(48)

Published: Aug. 22, 2024

Abstract Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O 2 ) concentrations. Here, we reprogrammed cellular hypoxia (low O signaling via gas tunnel engineering of prolyl hydroxylase (PHD2), a non‐heme iron dependent sensor. Using computational modeling protein techniques, identify critical residues therein that limit the flow PHD2’s catalytic core. We show systematic modification these can open constriction topology tunnel. kinetic stopped‐flow measurements with NO as surrogate diatomic gas, demonstrate up 3.5‐fold enhancement its association rate center tunnel‐engineered mutants. Our most effectively designed mutant displays 9‐fold enhanced efficiency ( k cat / K M =830±40 −1 s hydroxylating peptide mimic inducible transcription factor HIF‐1α, compared WT PHD2 =90±9 ). Furthermore, transfection plasmids express mutants HEK‐293T mammalian cells reveal significant reduction HIF‐1α downstream response transcripts under hypoxic conditions 1 % . Overall, studies highlight activation new pathway reprogram responses HIF cells.

Language: Английский

Citations

1
Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase DOI Creative Commons
Elizabeth R. Smithwick, Ambika Bhagi‐Damodaran, Anoop R. Damodaran

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 4, 2024

Determination of substrate binding affinity (

Language: Английский

Citations

0
Controllable multi-halogenation of a non-native substrate by SyrB2 iron halogenase DOI Creative Commons

R. Hunter Wilson,

Sourav Chatterjee, Elizabeth R. Smithwick

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: May 9, 2024

Geminal, multi-halogenated functional groups are widespread in natural products and pharmaceuticals, yet no synthetic methodologies exist that enable selective multi-halogenation of unactivated C-H bonds. Biocatalysts powerful tools for late-stage functionalization, as they operate with high degrees regio-, chemo-, stereoselectivity. 2-oxoglutarate (2OG)-dependent non-heme iron halogenases chlorinate brominate aliphatic bonds offering a solution achieving these challenging transformations. Here, we describe the ability halogenase, SyrB2, to controllably halogenate non-native substrate alpha-aminobutyric acid (Aba) yield mono-chlorinated, di-chlorinated, tri-chlorinated products. These chemoselective outcomes achieved by controlling loading 2OG cofactor SyrB2 biocatalyst. By using ferredoxin-based biological reductant electron transfer catalytic center demonstrate order-of-magnitude enhancement product were previously inaccessible any single halogenase enzyme. We also apply strategies broaden SyrB2's reactivity scope include multi-bromination chemoenzymatic conversion ethyl side chain Aba an ethylyne group. show how steric hindrance induced successive addition halogen atoms on Aba's C

Language: Английский

Citations

0
Dynamic metal coordination controls chemoselectivity in radical halogenases DOI
Elijah N. Kissman, Ioannis Kipouros, Jeffrey W. Slater

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 20, 2024

Abstract The activation of inert C( sp 3 )-H bonds by non-heme Fe enzymes plays a key role in metabolism, epigenetics, and signaling, while providing powerful biocatalytic platform for the chemical synthesis molecules with increased complexity. In this context, II /α-ketoglutarate-dependent radical halogenases represent broadly interesting system, as they are uniquely capable carrying out transfer diverse array bound anions following C-H activation. Here, we provide first experimental evidence that bifurcation H-atom abstraction rebound is driven both ability dynamic metal coordination sphere to reorganize well second-sphere hydrogen-bond network where only two residues (Asn224 Ile151) necessary sufficient. identification minimal motif provides paradigm understanding evolution catalytic plasticity these yields new insight into design principles which expand their reaction scope.

Language: Английский

Citations

0
Gas tunnel engineering of prolyl hydroxylase reprograms hypoxia signaling in cells DOI Creative Commons
Peter Windsor,

Haiping Ouyang,

Joseph A. G. da Costa

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Aug. 7, 2023

Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O

Language: Английский

Citations

0