J 2021

Computational Enzyme Stabilization Can Affect Folding Energy Landscapes and Lead to Catalytically Enhanced Domain-Swapped Dimers

MARKOVÁ, Klára, Antonín KUNKA, Klaudia CHMELOVÁ, Martin HAVLÁSEK, Petra BABKOVÁ et. al.

Basic information

Original name

Computational Enzyme Stabilization Can Affect Folding Energy Landscapes and Lead to Catalytically Enhanced Domain-Swapped Dimers

Authors

MARKOVÁ, Klára (203 Czech Republic, belonging to the institution), Antonín KUNKA (203 Czech Republic, belonging to the institution), Klaudia CHMELOVÁ (703 Slovakia, belonging to the institution), Martin HAVLÁSEK (203 Czech Republic, belonging to the institution), Petra BABKOVÁ (203 Czech Republic, belonging to the institution), Sérgio Manuel MARQUES (620 Portugal, belonging to the institution), Michal VAŠINA (203 Czech Republic, belonging to the institution), Joan PLANAS IGLESIAS (724 Spain, belonging to the institution), Radka CHALOUPKOVÁ (203 Czech Republic, belonging to the institution), David BEDNÁŘ (203 Czech Republic, belonging to the institution), Zbyněk PROKOP (203 Czech Republic, belonging to the institution), Jiří DAMBORSKÝ (203 Czech Republic, guarantor, belonging to the institution) and Martin MAREK (203 Czech Republic, belonging to the institution)

Edition

ACS Catalysis, WASHINGTON, AMER CHEMICAL SOC, 2021, 2155-5435

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10403 Physical chemistry

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

Impact factor

Impact factor: 13.700

RIV identification code

RIV/00216224:14310/21:00124052

Organization unit

Faculty of Science

DOI

UT WoS

000716773800006

Keywords in English

protein folding; protein design; alpha/beta-hydrolase; haloalkane dehalogenase; domain swapping; energy landscape; oligonicrization; catalytic efficiency; substrate inhibition

Tags

Tags

International impact, Reviewed
Změněno: 15/2/2023 23:13, Mgr. Michaela Hylsová, Ph.D.

Abstract

V originále

The functionality of an enzyme depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology applications. Here, we reveal a unique example where the computational stabilization of a monomeric alpha/beta-hydrolase enzyme (T-m = 73.5 degrees C; Delta T-m > 23 degrees C) affected the protein folding energy landscape. The introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded soluble domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations might (i) activate cryptic hinge-loop regions and (ii) establish secondary interfaces, where they make extensive noncovalent interactions between the intertwined protomers. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from small-angle X-ray scattering (SAXS) and cross-linking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers can be irreversibly converted into native-like monomers, suggesting that the domain swapping occurs exclusively in vivo. Crucially, the swapped-dimers exhibited advantageous catalytic properties such as an increased catalytic rate and elimination of substrate inhibition. These findings provide additional enzyme engineering avenues for next-generation biocatalysts.

Links

EF17_043/0009632, research and development project
Name: CETOCOEN Excellence
LM2015047, research and development project
Name: Česká národní infrastruktura pro biologická data (Acronym: ELIXIR-CZ)
Investor: Ministry of Education, Youth and Sports of the CR, Czech National Infrastructure for Biological Data
LM2018121, research and development project
Name: Výzkumná infrastruktura RECETOX (Acronym: RECETOX RI)
Investor: Ministry of Education, Youth and Sports of the CR, RECETOX RI
LM2018127, research and development project
Name: Česká infrastruktura pro integrativní strukturní biologii (Acronym: CIISB)
Investor: Ministry of Education, Youth and Sports of the CR
LM2018140, research and development project
Name: e-Infrastruktura CZ (Acronym: e-INFRA CZ)
Investor: Ministry of Education, Youth and Sports of the CR
MUNI/H/1561/2018, interní kód MU
Name: Decoding the molecular principles of enzyme evolution
Investor: Masaryk University, Individual High risk/high gain projects
792772, interní kód MU
Name: Structural and biochemical studies of an ancestral enzyme with dual dehalogenase and luciferase activity (Acronym: Ancestral)
Investor: European Union, MSCA Marie Skłodowska-Curie Actions (Excellent Science)
814418, interní kód MU
Name: Synthetic biology-guided engineering of Pseudomonas putida for biofluorination (Acronym: SinFonia)
Investor: European Union, Leadership in enabling and industrial technologies (LEIT) (Industrial Leadership)
857560, interní kód MU
(CEP code: EF17_043/0009632)
Name: CETOCOEN Excellence (Acronym: CETOCOEN Excellence)
Investor: European Union, Spreading excellence and widening participation