Azobenzene-Based Photoswitchable Substrates for Advanced
Mechanistic Studies of Model Haloalkane Dehalogenase Enzyme
Family

J 2024

Azobenzene-Based Photoswitchable Substrates for Advanced Mechanistic Studies of Model Haloalkane Dehalogenase Enzyme Family

SLÁNSKÁ, Michaela; Lenka ŠTACKOVÁ; Sérgio Manuel MARQUES; Peter ŠTACKO; Marek MARTÍNEK et al.

Základní údaje

Originální název

Azobenzene-Based Photoswitchable Substrates for Advanced Mechanistic Studies of Model Haloalkane Dehalogenase Enzyme Family

Autoři

SLÁNSKÁ, Michaela; Lenka ŠTACKOVÁ; Sérgio Manuel MARQUES; Peter ŠTACKO; Marek MARTÍNEK; Luboš JÍLEK; Martin TOUL; Jiří DAMBORSKÝ; David BEDNÁŘ; Petr KLÁN a Zbyněk PROKOP

Vydání

ACS Catalysis, WASHINGTON, AMER CHEMICAL SOC, 2024, 2155-5435

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10403 Physical chemistry

Stát vydavatele

Spojené státy

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 13.100

Označené pro přenos do RIV

Ano

Kód RIV

RIV/00216224:14310/24:00136913

Organizační jednotka

Přírodovědecká fakulta

Klíčová slova anglicky

photoswitch; azobezene; enzyme; transientkinetics; mechanism; haloalkane dehalogenase; time-resolved spectroscopy

Štítky

14313010, 14314010, 143180, rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 5. 6. 2025 10:23, Mgr. Marie Novosadová Šípková, DiS.

Anotace

V originále

The engineering of efficient enzymes for large-scale production of industrially relevant compounds is a challenging task. Utilizing rational protein design, which relies on a comprehensive understanding of mechanistic information, holds significant promise for achieving success in this endeavor. Pre-steady-state kinetic measurements, obtained either through fast-mixing techniques or photoswitchable substrates, provide crucial mechanistic insights. The latter approach not only furnishes mechanistic clarity but also affords real-time structural elucidation of reaction intermediates via time-resolved femtosecond crystallography. Unfortunately, only a limited number of such valuable mechanistic probes are available. To address this gap, we applied a multidisciplinary approach, including computational analysis, chemical synthesis, physicochemical property screening, and enzyme kinetics to identify promising candidates for photoswitchable probes. We demonstrate the approach by designing an azobenzene-based photoswitchable substrate tailored for haloalkane dehalogenases, a prototypic class of enzymes pivotal in developing computational tools for rational protein design. The probe was subjected to steady-state and pre-steady-state kinetic analysis, which revealed new insights about the catalytic behavior of the model biocatalysts. We employed laser-triggered Z-to-E azobenzene photoswitching to generate the productive isomer in situ, opening avenues for advanced mechanistic studies using time-resolved femtosecond crystallography. Our results not only pave the way for the mechanistic understanding of this model enzyme family, incorporating both kinetic and structural dimensions, but also propose a systematic approach to the rational design of photoswitchable enzymatic substrates.

Návaznosti

LM2018140, projekt VaV

Název: e-Infrastruktura CZ (Akronym: e-INFRA CZ)

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, e-Infrastruktura CZ

LM2023069, projekt VaV

Název: Výzkumná infrastruktura RECETOX

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Výzkumná infrastruktura RECETOX

857560, interní kód MU
(Kód CEP: EF17_043/0009632)

Název: CETOCOEN Excellence (Akronym: CETOCOEN Excellence)

Investor: Evropská unie, CETOCOEN Excellence, Spreading excellence and widening participation

90254, velká výzkumná infrastruktura

Název: e-INFRA CZ II

Přiložené soubory

2424817.pdf

Citovat

SLÁNSKÁ, Michaela; Lenka ŠTACKOVÁ; Sérgio Manuel MARQUES; Peter ŠTACKO; Marek MARTÍNEK; Luboš JÍLEK; Martin TOUL; Jiří DAMBORSKÝ; David BEDNÁŘ; Petr KLÁN a Zbyněk PROKOP. Azobenzene-Based Photoswitchable Substrates for Advanced Mechanistic Studies of Model Haloalkane Dehalogenase Enzyme Family. ACS Catalysis. WASHINGTON: AMER CHEMICAL SOC, 2024, roč. 14, č. 15, s. 11635-11645. ISSN 2155-5435. Dostupné z: https://doi.org/10.1021/acscatal.4c03503.

@article{2424817,
   author = {Slánská, Michaela and Štacková, Lenka and Marques, Sérgio Manuel and Štacko, Peter and Martínek, Marek and Jílek, Luboš and Toul, Martin and Damborský, Jiří and Bednář, David and Klán, Petr and Prokop, Zbyněk},
   article_location = {WASHINGTON},
   article_number = {15},
   doi = {https://doi.org/10.1021/acscatal.4c03503},
   keywords = {photoswitch; azobezene; enzyme; transientkinetics; mechanism; haloalkane dehalogenase; time-resolved spectroscopy},
   language = {eng},
   issn = {2155-5435},
   journal = {ACS Catalysis},
   note = {Nedoplňovat EF17_043/0009632.},
   title = {Azobenzene-Based Photoswitchable Substrates for Advanced Mechanistic Studies of Model Haloalkane Dehalogenase Enzyme Family},
   url = {https://pubs.acs.org/doi/10.1021/acscatal.4c03503},
   volume = {14},
   year = {2024}
}

TY  - JOUR
ID  - 2424817
AU  - Slánská, Michaela - Štacková, Lenka - Marques, Sérgio Manuel - Štacko, Peter - Martínek, Marek - Jílek, Luboš - Toul, Martin - Damborský, Jiří - Bednář, David - Klán, Petr - Prokop, Zbyněk
PY  - 2024
TI  - Azobenzene-Based Photoswitchable Substrates for Advanced Mechanistic Studies of Model Haloalkane Dehalogenase Enzyme Family
JF  - ACS Catalysis
VL  - 14
IS  - 15
SP  - 11635-11645
EP  - 11635-11645
PB  - AMER CHEMICAL SOC
SN  - 21555435
N1  - Nedoplňovat EF17_043/0009632.
KW  - photoswitch
KW  - azobezene
KW  - enzyme
KW  - transientkinetics
KW  - mechanism
KW  - haloalkane dehalogenase
KW  - time-resolved spectroscopy
UR  - https://pubs.acs.org/doi/10.1021/acscatal.4c03503
N2  - The engineering of efficient enzymes for large-scale production of industrially relevant compounds is a challenging task. Utilizing rational protein design, which relies on a comprehensive understanding of mechanistic information, holds significant promise for achieving success in this endeavor. Pre-steady-state kinetic measurements, obtained either through fast-mixing techniques or photoswitchable substrates, provide crucial mechanistic insights. The latter approach not only furnishes mechanistic clarity but also affords real-time structural elucidation of reaction intermediates via time-resolved femtosecond crystallography. Unfortunately, only a limited number of such valuable mechanistic probes are available. To address this gap, we applied a multidisciplinary approach, including computational analysis, chemical synthesis, physicochemical property screening, and enzyme kinetics to identify promising candidates for photoswitchable probes. We demonstrate the approach by designing an azobenzene-based photoswitchable substrate tailored for haloalkane dehalogenases, a prototypic class of enzymes pivotal in developing computational tools for rational protein design. The probe was subjected to steady-state and pre-steady-state kinetic analysis, which revealed new insights about the catalytic behavior of the model biocatalysts. We employed laser-triggered Z-to-E azobenzene photoswitching to generate the productive isomer in situ, opening avenues for advanced mechanistic studies using time-resolved femtosecond crystallography. Our results not only pave the way for the mechanistic understanding of this model enzyme family, incorporating both kinetic and structural dimensions, but also propose a systematic approach to the rational design of photoswitchable enzymatic substrates.
ER  -

SLÁNSKÁ, Michaela; Lenka ŠTACKOVÁ; Sérgio Manuel MARQUES; Peter ŠTACKO; Marek MARTÍNEK; Luboš JÍLEK; Martin TOUL; Jiří DAMBORSKÝ; David BEDNÁŘ; Petr KLÁN a Zbyněk PROKOP. Azobenzene-Based Photoswitchable Substrates for Advanced Mechanistic Studies of Model Haloalkane Dehalogenase Enzyme Family. \textit{ACS Catalysis}. WASHINGTON: AMER CHEMICAL SOC, 2024, roč.~14, č.~15, s.~11635-11645. ISSN~2155-5435. Dostupné z: https://doi.org/10.1021/acscatal.4c03503.

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