Substrate Anchoring and Flexibility Reduction in CYP153A(M.aq) Leads to Highly Improved Efficiency toward Octanoic Acid

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00159816%3A_____%2F21%3A00075254" target="_blank" >RIV/00159816:_____/21:00075254 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216224:14310/21:00122271

Výsledek na webu

<a href="https://pubs.acs.org/doi/10.1021/acscatal.0c05193" target="_blank" >https://pubs.acs.org/doi/10.1021/acscatal.0c05193</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1021/acscatal.0c05193" target="_blank" >10.1021/acscatal.0c05193</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Substrate Anchoring and Flexibility Reduction in CYP153A(M.aq) Leads to Highly Improved Efficiency toward Octanoic Acid

Popis výsledku v původním jazyce

Cytochrome P450 CYP153A(M.aq) from Marinobacter aquaeolei serves as a model enzyme for the terminal (omega-) hydroxylation of medium- to long-chain fatty acids. We have engineered this enzyme using different mutagenesis approaches based on structure-sequence-alignments within the 3DM database and crystal structures of CYP153A(M.aq) and a homologue CYP153A(P.sp). Applying these focused mutagenesis strategies and site-directed saturation mutagenesis, we created a variant that omega-hydroxylates octanoic acid. The M.aqRLT variant exhibited 151-fold improved catalytic efficiency and showed strongly improved substrate binding (25-fold reduced K-m compared to the wild type). We then used molecular dynamics simulations to gain deeper insights into the dynamics of the protein. We found the tunnel modifications and the two loop regions showing greatly reduced flexibility in the engineered variant were the main features responsible for stabilizing the enzyme-substrate complex and enhancing the catalytic efficiency. Additionally, we showed that a previously known fatty acid anchor (Q129R) interacts significantly with the ligand to hold it in the reactive position, thereby boosting the activity of the variant M.aqRLT toward octanoic acid. The study demonstrates the significant effects of both substrate stabilization and the impact of enzyme flexibility on catalytic efficiency. These results could guide the future engineering of enzymes with deeply buried active sites to increase or even establish activities toward yet unknown types of substrates.

Název v anglickém jazyce

Substrate Anchoring and Flexibility Reduction in CYP153A(M.aq) Leads to Highly Improved Efficiency toward Octanoic Acid

Popis výsledku anglicky

Cytochrome P450 CYP153A(M.aq) from Marinobacter aquaeolei serves as a model enzyme for the terminal (omega-) hydroxylation of medium- to long-chain fatty acids. We have engineered this enzyme using different mutagenesis approaches based on structure-sequence-alignments within the 3DM database and crystal structures of CYP153A(M.aq) and a homologue CYP153A(P.sp). Applying these focused mutagenesis strategies and site-directed saturation mutagenesis, we created a variant that omega-hydroxylates octanoic acid. The M.aqRLT variant exhibited 151-fold improved catalytic efficiency and showed strongly improved substrate binding (25-fold reduced K-m compared to the wild type). We then used molecular dynamics simulations to gain deeper insights into the dynamics of the protein. We found the tunnel modifications and the two loop regions showing greatly reduced flexibility in the engineered variant were the main features responsible for stabilizing the enzyme-substrate complex and enhancing the catalytic efficiency. Additionally, we showed that a previously known fatty acid anchor (Q129R) interacts significantly with the ligand to hold it in the reactive position, thereby boosting the activity of the variant M.aqRLT toward octanoic acid. The study demonstrates the significant effects of both substrate stabilization and the impact of enzyme flexibility on catalytic efficiency. These results could guide the future engineering of enzymes with deeply buried active sites to increase or even establish activities toward yet unknown types of substrates.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10403 - Physical chemistry

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

2021

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

11

Číslo periodika v rámci svazku

5

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

8

Strana od-do

3182-3189

Kód UT WoS článku

000626844200065

EID výsledku v databázi Scopus

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