| RAPP, Lea R., Sérgio Manuel MARQUES, Erna ZUKIC, Benjamin ROWLINSON, Mahima SHARMA, Gideon GROGAN, Jiří DAMBORSKÝ and 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, vol. 11, No 5, p. 3182-3189. ISSN 2155-5435. Available from: https://dx.doi.org/10.1021/acscatal.0c05193. |
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@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 = {http://dx.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 and 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, vol.~11, No~5, p.~3182-3189. ISSN~2155-5435. Available from: https://dx.doi.org/10.1021/acscatal.0c05193.
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