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

Result code in 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>

Alternative codes found

RIV/00216224:14310/21:00122271

Result on the web

<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>

Result language

angličtina

Original language name

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

Original language description

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.

Czech name

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Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

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OECD FORD branch

10403 - Physical chemistry

Project

Result was created during the realization of more than one project. More information in the Projects tab.

Continuities

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Publication year

2021

Confidentiality

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

Name of the periodical

ACS Catalysis

ISSN

2155-5435

e-ISSN

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Volume of the periodical

11

Issue of the periodical within the volume

5

Country of publishing house

US - UNITED STATES

Number of pages

8

Pages from-to

3182-3189

UT code for WoS article

000626844200065

EID of the result in the Scopus database

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