Light-Emitting Dehalogenases: Reconstruction of Multifunctional Biocatalysts

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00209805%3A_____%2F19%3A00078138" target="_blank" >RIV/00209805:_____/19:00078138 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216224:14310/19:00107511 RIV/00159816:_____/19:00071064

Výsledek na webu

<a href="https://pubs.acs.org/doi/full/10.1021/acscatal.9b01031" target="_blank" >https://pubs.acs.org/doi/full/10.1021/acscatal.9b01031</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1021/acscatal.9b01031" target="_blank" >10.1021/acscatal.9b01031</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Light-Emitting Dehalogenases: Reconstruction of Multifunctional Biocatalysts

Popis výsledku v původním jazyce

To obtain structural insights into the emergence of biological functions from catalytically promiscuous enzymes, we reconstructed an ancestor of catalytically distinct, but evolutionarily related, haloalkane dehalogenases (EC 3.8.1.5) and Renilla luciferase (EC 1.13.12.5). This ancestor has both hydrolase and monooxygenase activities. Its crystal structure solved to 1.39 Å resolution revealed the presence of a catalytic pentad conserved in both dehalogenase and luciferase descendants and a molecular oxygen bound in between two residues typically stabilizing a halogen anion. The differences in the conformational dynamics of the specificity-determining cap domains between the ancestral and descendant enzymes were accessed by molecular dynamics and hydrogenMINUS SIGN deuterium exchange mass spectrometry. Stopped-flow analysis revealed that the alkyl enzyme intermediate formed in the luciferase-catalyzed reaction is trapped by blockage of a hydrolytic reaction step. A single-point mutation (Ala54Pro) adjacent to one of the catalytic residues bestowed hydrolase activity on the modern luciferase by enabling cleavage of this intermediate. Thus, a single substitution next to the catalytic pentad may enable the emergence of promiscuous activity at the enzyme class level, and ancestral reconstruction has a clear potential for obtaining multifunctional catalysts.

Název v anglickém jazyce

Light-Emitting Dehalogenases: Reconstruction of Multifunctional Biocatalysts

Popis výsledku anglicky

To obtain structural insights into the emergence of biological functions from catalytically promiscuous enzymes, we reconstructed an ancestor of catalytically distinct, but evolutionarily related, haloalkane dehalogenases (EC 3.8.1.5) and Renilla luciferase (EC 1.13.12.5). This ancestor has both hydrolase and monooxygenase activities. Its crystal structure solved to 1.39 Å resolution revealed the presence of a catalytic pentad conserved in both dehalogenase and luciferase descendants and a molecular oxygen bound in between two residues typically stabilizing a halogen anion. The differences in the conformational dynamics of the specificity-determining cap domains between the ancestral and descendant enzymes were accessed by molecular dynamics and hydrogenMINUS SIGN deuterium exchange mass spectrometry. Stopped-flow analysis revealed that the alkyl enzyme intermediate formed in the luciferase-catalyzed reaction is trapped by blockage of a hydrolytic reaction step. A single-point mutation (Ala54Pro) adjacent to one of the catalytic residues bestowed hydrolase activity on the modern luciferase by enabling cleavage of this intermediate. Thus, a single substitution next to the catalytic pentad may enable the emergence of promiscuous activity at the enzyme class level, and ancestral reconstruction has a clear potential for obtaining multifunctional catalysts.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2019

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

ACS catalysis

ISSN

2155-5435

e-ISSN

—

Svazek periodika

2019

Číslo periodika v rámci svazku

9

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

14

Strana od-do

"4810−4823"

Kód UT WoS článku

000471212600009

EID výsledku v databázi Scopus

2-s2.0-85065584281