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

J 2021

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

RAPP, Lea R.; Sérgio Manuel MARQUES; Erna ZUKIC; Benjamin ROWLINSON; Mahima SHARMA et al.

Základní údaje

Originální název

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

Autoři

RAPP, Lea R.; Sérgio Manuel MARQUES; Erna ZUKIC; Benjamin ROWLINSON; Mahima SHARMA; Gideon GROGAN; Jiří DAMBORSKÝ a Bernhard HAUER

Vydání

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

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10403 Physical chemistry

Stát vydavatele

Spojené státy

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 13.700

Označené pro přenos do RIV

Ano

Kód RIV

RIV/00216224:14310/21:00122271

Organizační jednotka

Přírodovědecká fakulta

Klíčová slova anglicky

biocatalysis; enzyme engineering; molecular dynamics; computational chemistry; cytochrome P450

Štítky

14314010, 143180, rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 16. 2. 2023 12:40, Mgr. Michaela Hylsová, Ph.D.

Anotace

V originále

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.

Návaznosti

EF17_043/0009632, projekt VaV

Název: CETOCOEN Excellence

LM2018140, projekt VaV

Název: e-Infrastruktura CZ (Akronym: e-INFRA CZ)

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, e-Infrastruktura CZ

Citovat

RAPP, Lea R.; Sérgio Manuel MARQUES; Erna ZUKIC; Benjamin ROWLINSON; Mahima SHARMA; Gideon GROGAN; Jiří DAMBORSKÝ a Bernhard HAUER. Substrate Anchoring and Flexibility Reduction in CYP153A(M.aq) Leads to Highly Improved Efficiency toward Octanoic Acid. ACS Catalysis. WASHINGTON: AMER CHEMICAL SOC, 2021, roč. 11, č. 5, s. 3182-3189. ISSN 2155-5435. Dostupné z: https://doi.org/10.1021/acscatal.0c05193.

@article{1790205,
   author = {Rapp, Lea R. and Marques, Sérgio Manuel and Zukic, Erna and Rowlinson, Benjamin and Sharma, Mahima and Grogan, Gideon and Damborský, Jiří and Hauer, Bernhard},
   article_location = {WASHINGTON},
   article_number = {5},
   doi = {https://doi.org/10.1021/acscatal.0c05193},
   keywords = {biocatalysis; enzyme engineering; molecular dynamics; computational chemistry; cytochrome P450},
   language = {eng},
   issn = {2155-5435},
   journal = {ACS Catalysis},
   title = {Substrate Anchoring and Flexibility Reduction in CYP153A(M.aq) Leads to Highly Improved Efficiency toward Octanoic Acid},
   url = {https://pubs.acs.org/doi/10.1021/acscatal.0c05193},
   volume = {11},
   year = {2021}
}

TY  - JOUR
ID  - 1790205
AU  - Rapp, Lea R. - Marques, Sérgio Manuel - Zukic, Erna - Rowlinson, Benjamin - Sharma, Mahima - Grogan, Gideon - Damborský, Jiří - Hauer, Bernhard
PY  - 2021
TI  - Substrate Anchoring and Flexibility Reduction in CYP153A(M.aq) Leads to Highly Improved Efficiency toward Octanoic Acid
JF  - ACS Catalysis
VL  - 11
IS  - 5
SP  - 3182-3189
EP  - 3182-3189
PB  - AMER CHEMICAL SOC
SN  - 21555435
KW  - biocatalysis
KW  - enzyme engineering
KW  - molecular dynamics
KW  - computational chemistry
KW  - cytochrome P450
UR  - https://pubs.acs.org/doi/10.1021/acscatal.0c05193
N2  - 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.
ER  -

RAPP, Lea R.; Sérgio Manuel MARQUES; Erna ZUKIC; Benjamin ROWLINSON; Mahima SHARMA; Gideon GROGAN; Jiří DAMBORSKÝ a Bernhard HAUER. Substrate Anchoring and Flexibility Reduction in CYP153A(M.aq) Leads to Highly Improved Efficiency toward Octanoic Acid. \textit{ACS Catalysis}. WASHINGTON: AMER CHEMICAL SOC, 2021, roč.~11, č.~5, s.~3182-3189. ISSN~2155-5435. Dostupné z: https://doi.org/10.1021/acscatal.0c05193.

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