Structural and catalytic effects of surface loop-helix transplantation within haloalkane dehalogenase family

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60076658%3A12310%2F20%3A43902332" target="_blank" >RIV/60076658:12310/20:43902332 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/68378050:_____/20:00539596 RIV/00216224:14310/20:00114750

Výsledek na webu

<a href="https://www.sciencedirect.com/science/article/pii/S2001037020302828?via%3Dihub" target="_blank" >https://www.sciencedirect.com/science/article/pii/S2001037020302828?via%3Dihub</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.csbj.2020.05.019" target="_blank" >10.1016/j.csbj.2020.05.019</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Structural and catalytic effects of surface loop-helix transplantation within haloalkane dehalogenase family

Popis výsledku v původním jazyce

Engineering enzyme catalytic properties is important for basic research as well as for biotechnological applications. We have previously shown that the reshaping of enzyme access tunnels via the deletion of a short surface loop element may yield a haloalkane dehalogenase variant with markedly modified substrate specificity and enantioselectivity. Here, we conversely probed the effects of surface loop-helix transplantation from one enzyme to another within the enzyme family of haloalkane dehalogenases. Precisely, we transplanted a nine-residue long extension of L9 loop and alpha 4 helix from DbjA into the corresponding site of DbeA. Biophysical characterization showed that this fragment transplantation did not affect the overall protein fold or oligomeric state, but lowered protein stability (Delta T-m = -5 to 6 degrees C). Interestingly, the crystal structure of DbeA mutant revealed the unique structural features of enzyme access tunnels, which are known determinants of catalytic properties for this enzyme family. Biochemical data confirmed that insertion increased activity of DbeA with various halogenated substrates and altered its enantioselectivity with several linear beta-bromoalkanes. Our findings support a protein engineering strategy employing surface loop-helix transplantation for construction of novel protein catalysts with modified catalytic properties. (C) 2020 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.

Název v anglickém jazyce

Structural and catalytic effects of surface loop-helix transplantation within haloalkane dehalogenase family

Popis výsledku anglicky

Engineering enzyme catalytic properties is important for basic research as well as for biotechnological applications. We have previously shown that the reshaping of enzyme access tunnels via the deletion of a short surface loop element may yield a haloalkane dehalogenase variant with markedly modified substrate specificity and enantioselectivity. Here, we conversely probed the effects of surface loop-helix transplantation from one enzyme to another within the enzyme family of haloalkane dehalogenases. Precisely, we transplanted a nine-residue long extension of L9 loop and alpha 4 helix from DbjA into the corresponding site of DbeA. Biophysical characterization showed that this fragment transplantation did not affect the overall protein fold or oligomeric state, but lowered protein stability (Delta T-m = -5 to 6 degrees C). Interestingly, the crystal structure of DbeA mutant revealed the unique structural features of enzyme access tunnels, which are known determinants of catalytic properties for this enzyme family. Biochemical data confirmed that insertion increased activity of DbeA with various halogenated substrates and altered its enantioselectivity with several linear beta-bromoalkanes. Our findings support a protein engineering strategy employing surface loop-helix transplantation for construction of novel protein catalysts with modified catalytic properties. (C) 2020 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10610 - Biophysics

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)

Rok uplatnění

2020

Kód důvěrnosti údajů

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

Název periodika

Computational and Structural Biotechnology Journal

ISSN

2001-0370

e-ISSN

—

Svazek periodika

18

Číslo periodika v rámci svazku

2020

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

11

Strana od-do

1352-1362

Kód UT WoS článku

000607350300011

EID výsledku v databázi Scopus

2-s2.0-85086588894