Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering
| Autor: | Wim J. Quax, Ronald van Merkerk, Esmée Klumpenaar, Ingy I Abdallah |
|---|---|
| Přispěvatelé: | Groningen Research Institute of Pharmacy, Chemical and Pharmaceutical Biology, Molecular Microbiology, Nanotechnology and Biophysics in Medicine (NANOBIOMED), Biopharmaceuticals, Discovery, Design and Delivery (BDDD) |
| Rok vydání: | 2018 |
| Předmět: |
amorpha-4
0301 basic medicine transferase Farnesyl pyrophosphate lcsh:Medicine Protein Engineering Pyrophosphate 11-diene synthase chemistry.chemical_compound Catalytic Domain Transferase genetics amorpha-4 11-diene lcsh:Science Magnesium ion Multidisciplinary biology mass fragmentography Recombinant Proteins Diphosphates enzyme active site pyrophosphoric acid derivative Sesquiterpenes Amorpha-4 11-diene sesquiterpene Stereochemistry 11-diene chemistry Catalysis Gas Chromatography-Mass Spectrometry Article 03 medical and health sciences site directed mutagenesis Peptide Library Escherichia coli Enzyme kinetics procedures Polycyclic Sesquiterpenes Alkyl and Aryl Transferases diphosphoric acid lcsh:R Active site Protein engineering 030104 developmental biology biology.protein Mutagenesis Site-Directed lcsh:Q amorpha-4 11-diene synthase metabolism recombinant protein |
| Zdroj: | Scientific Reports Scientific Reports, Vol 8, Iss 1, Pp 1-11 (2018) Scientific Reports, 8(1):9961. Nature Publishing Group |
| ISSN: | 2045-2322 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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