Publication

Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering

Abdallah, I. I., van Merkerk, R., Klumpenaar, E. & Quax, W. J., 2-Jul-2018, In : Scientific Reports. 8, 1, 1 p., 9961.

Research output: Contribution to journalArticleAcademicpeer-review

APA

Abdallah, I. I., van Merkerk, R., Klumpenaar, E., & Quax, W. J. (2018). Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering. Scientific Reports, 8(1), [9961]. https://doi.org/10.1038/s41598-018-28177-4

Author

Abdallah, Ingy I ; van Merkerk, Ronald ; Klumpenaar, Esmée ; Quax, Wim J. / Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering. In: Scientific Reports. 2018 ; Vol. 8, No. 1.

Harvard

Abdallah, II, van Merkerk, R, Klumpenaar, E & Quax, WJ 2018, 'Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering', Scientific Reports, vol. 8, no. 1, 9961. https://doi.org/10.1038/s41598-018-28177-4

Standard

Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering. / Abdallah, Ingy I; van Merkerk, Ronald; Klumpenaar, Esmée; Quax, Wim J.

In: Scientific Reports, Vol. 8, No. 1, 9961, 02.07.2018.

Research output: Contribution to journalArticleAcademicpeer-review

Vancouver

Abdallah II, van Merkerk R, Klumpenaar E, Quax WJ. Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering. Scientific Reports. 2018 Jul 2;8(1). 9961. https://doi.org/10.1038/s41598-018-28177-4


BibTeX

@article{6ca06b8ec5764845ac0f15ebf8f6a230,
title = "Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering",
abstract = "Amorpha-4,11-diene synthase (ADS) cyclizes the substrate farnesyl pyrophosphate to produce amorpha-4,11-diene as a major product. This is considered the first committed and rate-limiting step in the biosynthesis of the antimalarial artemisinin. Here, we utilize a reported 3D model of ADS to perform mutability landscape guided enzyme engineering. A mutant library of 258 variants along sixteen active site residues was created then screened for catalytic activity and product profile. This allowed for identification of the role of some of these residues in the mechanism. R262 constrains the released pyrophosphate group along with magnesium ions. The aromatic residues (W271, Y519 and F525) stabilize the intermediate carbocations while T296, G400, G439 and L515 help with the 1,6- and 1,10-ring closures. Finally, W271 is suggested to act as active site base along with T399, which ensures regioselective deprotonation. The mutability landscape also helped determine variants with improved catalytic activity. H448A showed ~4 fold increase in catalytic efficiency and the double mutation T399S/H448A improved kcat by 5 times. This variant can be used to enhance amorphadiene production and in turn artemisinin biosynthesis. Our findings provide the basis for the first step in improving industrial production of artemisinin and they open up possibilities for further engineering and understanding of ADS.",
keywords = "amorpha-4,11-diene, amorpha-4,11-diene synthase, diphosphoric acid, pyrophosphoric acid derivative, recombinant protein, sesquiterpene, transferase, catalysis, chemistry, enzyme active site, Escherichia coli, genetics, mass fragmentography, metabolism, peptide library, procedures, protein engineering, site directed mutagenesis",
author = "Abdallah, {Ingy I} and {van Merkerk}, Ronald and Esm{\'e}e Klumpenaar and Quax, {Wim J}",
year = "2018",
month = "7",
day = "2",
doi = "10.1038/s41598-018-28177-4",
language = "English",
volume = "8",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",
number = "1",

}

RIS

TY - JOUR

T1 - Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering

AU - Abdallah, Ingy I

AU - van Merkerk, Ronald

AU - Klumpenaar, Esmée

AU - Quax, Wim J

PY - 2018/7/2

Y1 - 2018/7/2

N2 - Amorpha-4,11-diene synthase (ADS) cyclizes the substrate farnesyl pyrophosphate to produce amorpha-4,11-diene as a major product. This is considered the first committed and rate-limiting step in the biosynthesis of the antimalarial artemisinin. Here, we utilize a reported 3D model of ADS to perform mutability landscape guided enzyme engineering. A mutant library of 258 variants along sixteen active site residues was created then screened for catalytic activity and product profile. This allowed for identification of the role of some of these residues in the mechanism. R262 constrains the released pyrophosphate group along with magnesium ions. The aromatic residues (W271, Y519 and F525) stabilize the intermediate carbocations while T296, G400, G439 and L515 help with the 1,6- and 1,10-ring closures. Finally, W271 is suggested to act as active site base along with T399, which ensures regioselective deprotonation. The mutability landscape also helped determine variants with improved catalytic activity. H448A showed ~4 fold increase in catalytic efficiency and the double mutation T399S/H448A improved kcat by 5 times. This variant can be used to enhance amorphadiene production and in turn artemisinin biosynthesis. Our findings provide the basis for the first step in improving industrial production of artemisinin and they open up possibilities for further engineering and understanding of ADS.

AB - Amorpha-4,11-diene synthase (ADS) cyclizes the substrate farnesyl pyrophosphate to produce amorpha-4,11-diene as a major product. This is considered the first committed and rate-limiting step in the biosynthesis of the antimalarial artemisinin. Here, we utilize a reported 3D model of ADS to perform mutability landscape guided enzyme engineering. A mutant library of 258 variants along sixteen active site residues was created then screened for catalytic activity and product profile. This allowed for identification of the role of some of these residues in the mechanism. R262 constrains the released pyrophosphate group along with magnesium ions. The aromatic residues (W271, Y519 and F525) stabilize the intermediate carbocations while T296, G400, G439 and L515 help with the 1,6- and 1,10-ring closures. Finally, W271 is suggested to act as active site base along with T399, which ensures regioselective deprotonation. The mutability landscape also helped determine variants with improved catalytic activity. H448A showed ~4 fold increase in catalytic efficiency and the double mutation T399S/H448A improved kcat by 5 times. This variant can be used to enhance amorphadiene production and in turn artemisinin biosynthesis. Our findings provide the basis for the first step in improving industrial production of artemisinin and they open up possibilities for further engineering and understanding of ADS.

KW - amorpha-4,11-diene

KW - amorpha-4,11-diene synthase

KW - diphosphoric acid

KW - pyrophosphoric acid derivative

KW - recombinant protein

KW - sesquiterpene

KW - transferase

KW - catalysis

KW - chemistry

KW - enzyme active site

KW - Escherichia coli

KW - genetics

KW - mass fragmentography

KW - metabolism

KW - peptide library

KW - procedures

KW - protein engineering

KW - site directed mutagenesis

U2 - 10.1038/s41598-018-28177-4

DO - 10.1038/s41598-018-28177-4

M3 - Article

C2 - 29967474

VL - 8

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

IS - 1

M1 - 9961

ER -

ID: 62493494