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.
Highlights
Amorpha-4,11-diene synthase (ADS) is an enzyme attracting world-wide interest as it is a sesquiterpene synthase that catalyzes the conversion of farnesyl pyrophosphate (FPP) to amorpha-4,11-diene, which is the precursor for the important antimalarial artemisinin
We selected sixteen active site residues to create a mutability landscape of ADS. This mutability landscape is screened for catalytic activity and product profile to determine the residues involved in the mechanism of ADS and to select variants with enhanced catalytic activity compared to the wild type with the aim of improving the overall production of artemisinin
A mutant library of the sixteen active site residues was generated showing high diversity of amino acids and their locations in the active site. With this library we have identified residues involved in the catalytic mechanism and obtained variants with improved activity compared to the wild type
Summary
Amorpha-4,11-diene synthase (ADS) is an enzyme attracting world-wide interest as it is a sesquiterpene synthase that catalyzes the conversion of farnesyl pyrophosphate (FPP) to amorpha-4,11-diene, which is the precursor for the important antimalarial artemisinin. The generation of mutability landscapes where a large number of protein variants are screened to discover the effect of each single amino acid substitution on enzyme activity, stability, and/or, selectivity is a valuable tool of protein engineering[16] These landscapes produce detailed maps of favorable, neutral, and detrimental amino acids for each residue position in relation to different www.nature.com/scientificreports/. We selected sixteen active site residues to create a mutability landscape of ADS This mutability landscape is screened for catalytic activity and product profile to determine the residues involved in the mechanism of ADS and to select variants with enhanced catalytic activity compared to the wild type with the aim of improving the overall production of artemisinin. As a proof of concept, in E. coli the amount of amorphadiene produced by the highly active variants was compared to that of the wild type ADS
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