Systematic identification of functional residues of Artemisia annua amorpha-4,11-diene synthase

Abstract

Terpene synthases (TPSs) are responsible for the extremely diversified and complex structure of terpenoids. Amorpha-4,11-diene synthase (ADS) has a high (90%) fidelity in generating the sesquiterpene precursor for the biosynthesis of artemisinin, an antimalarial drug, however, little is known about how active site residues of ADS are involved in carbocation rearrangement and cyclization reactions. Here, we identify seven residues that are key to most of the catalytic steps in ADS. By structural modeling and amino acid sequence alignments of ADS with two functionally relevant sesquiterpene synthases from , we performed site-directed mutagenesis and found that a single substitution, T296V, impaired the ring closure activity almost completely, and tetra-substitutions (L374Y/L404V/L405I/G439S) led to an enzyme generating 80% monocyclic bisabolyl-type sesquiterpenes, whereas a double mutant (T399L/T447G) showed compromised activity in regioselective deprotonation to yield 34.7 and 37.7% normal and aberrant deprotonation products, respectively. Notably, Thr296, Leu374, Gly439, Thr399, and Thr447, which play a major role in directing catalytic cascades, are located around conserved metal-binding motifs and function through impacting the folding of the substrate/intermediate, implying that residues surrounding the two motifs could be valuable targets for engineering TPS activity. Using this knowledge, we substantially increased amorpha-4,11-diene production in a near-additive manner by engineering Thr399 and Thr447 for product release. Our results provide new insight for the rational design of enzyme activity using synthetic biology.

In planta and in silico characterization of five sesquiterpene synthases from Vitis vinifera (cv. Shiraz) berries

“…Further, mutants in which residue Leu 374 (Tyr 376 in TEAS) in helix F is substituted by larger residues (e.g. Tyr or Phe), the 1,10-ring closure of the bisabolyl cation (which is the second cyclization event in ADS) is moderately impaired and the mutants have aberrant product profiles, some being 1,11-ring closure products (Fang et al 2017). Tyr is the corresponding residue all the investigated VvShirazTPS enzymes (Fig.…”

“…Site-directed mutagenesis of Thr 296 (Ser 298 in TEAS) in an amorpha-4,11-diene synthase (ADS) from Artemisia annua iterates the importance of this residue in the cyclization reaction, since substituting it with Val produces profiles of mainly linear products instead of cyclic (Fang et al 2017). The equivalent residue is serine for all of the investigated VvShirazTPS enzymes.…”

“…Substituting the Gly 402 with Asp or Cys led to an even greater increase in the 1,11-cyclization, whereas more bulky residues started to decrease the 1,11-cyclization activity (Thr and Val) or abolished it altogether (Leu, Ile, and Phe) (Gonzalez et al 2014). The Thr residue of this motif in ADS plays a crucial role in the deprotonation of the 12/13 proton of amorphadien-11-yl cation (Li et al 2013;Fang et al 2017). In all the VvShirazTPS enzymes investigated in our study, Thr and Ser were the corresponding residues of Thr 401 and Thr 402 , respectively, whereas Thr 403 is either Ala (VvShirazTPS-Y1 and VvGuaS) or Gly (in all the others).…”

“…For instance, changing Thr to Ser in VvGauS did not have large implications on an aberrant product formation (Drew et al 2016). However, a change like this could have an impact on the catalytic efficiency (Fang et al 2017). In ZmTPS4 and ZmTPS5 from Z. mays, Ala 409 and Gly 409 correspond to Thr 402 in TEAS, respectively.…”

An estimate is worth about a thousand experiments: using order-of-magnitude estimates to identify cellular engineering targets

“…We show a rough estimate to justify, in retrospect, this effort. The enzyme amorphadiene synthase (ADS) from Artemisia annua catalyzes the cyclization of farnesyl diphosphate to amorpha-4,11-diene a turnover number of 0.2 s −1 [6], which is relatively slow compared to the other enzymes in the pathway [7] (Table 1). …”