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

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.

Original languageEnglish
Article number9961
Number of pages1
JournalScientific Reports
Volume8
Issue number1
Publication statusPublished - 2-Jul-2018

    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

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