Redesigning the Molecular Choreography to Prevent Hydroxylation in Germacradien-11-ol Synthase Catalysis

Abstract

Natural sesquiterpene synthases have evolved to make complex terpenoids by quenching reactive carbocations either by proton transfer or by hydroxylation (water capture), depending on their active site. Germacradien-11-ol synthase (Gd11olS) from Streptomyces coelicolor catalyzes the cyclization of farnesyl diphosphate (FDP) into the hydroxylated sesquiterpene germacradien-11-ol. Here, we combine experiment and simulation to guide the redesign of its active site pocket to avoid hydroxylation of the product. Molecular dynamics simulations indicate two regions between which water molecules can flow that are responsible for hydroxylation. Point mutations of selected residues result in variants that predominantly form a complex nonhydroxylated product, which we identify as isolepidozene. Our results indicate how these mutations subtly change the molecular choreography in the Gd11olS active site and thereby pave the way for the engineering of terpene synthases to make complex terpenoid products.

Mechanistic Investigations on Microbial Type I Terpene Synthases through Site-Directed Mutagenesis

“…Especially, the mutation of H320 gave pronounced effects with significantly reduced activity and a product shift towards isolepidozene ( 10 , Scheme 5 ). 32 In the corresponding position of EIZS a Phe residue is found. Its exchange (F332A) showed a strongly reduced catalytic activity with formation of 37% 19 (Scheme 6 ).…”

“…14 For Gd11olS, similar observations were made with G188A yielding the neutral intermediate 11 with strongly reduced activity, while G188V was inactive. 32 Thus, in both cases, exchange of the effector against Ala seems to prevent reprotonation of the neutral intermediate, while exchange against Val completely disrupts enzyme activity. Also for sestermobaraene synthase, exchange of the effector (G184L) resulted in abolished production.…”

“…For germacradien-11-ol synthase (Gd11olS), the N-terminal domain of geosmin synthase from Streptomyces coelicolor, the linker exchange V187N yielded the stable intermediate isolepidozene (10), while V187A gave moderately reduced activity with formation of germacradien-11-ol (11), and V187D was inactive (Scheme 5). 32 Exchange of the effector in SdS against a small residue (G182A) gave slightly reduced activity, with accumulation of the neutral intermediate 8, while larger residues (G182V, G182P) resulted in inactivity. 14 For Gd11olS, similar observations were made with G188A yielding the neutral intermediate 11 with strongly reduced activity, while G188V was inactive.…”

“…42 For CotB2, the F107A and F107G variants resulted in the formation of cembrene A (21), while the F107Y exchange yielded cyclooctat-1,7-diene (26) and another unidentified diterpene, 43 and F107L gave, besides 28, several new compounds including 3,7-dolabelladiene-9-ol ( 24) and cyclooctat-6-en-8-ol (27). 44 A recently characterised multiproduct diterpene synthase from Cryptosporangium polytrichastri (CpPS) catalyses conversion of GGPP into the main product polytrichas-trene A (29), besides polytrichastrol A (30), wanju-2,5-diene (31), wanju-2,6-diene (32) and thunbergol (33, Figure 10). In this enzyme the positions corresponding to L78 and F79 in SdS are occupied by small residues (G90 and A91).…”

Predictive Engineering of Class I Terpene Synthases Using Experimental and Computational Approaches

“…A recent study on the STS germacradien‐11‐ol synthase revealed that subtle changes in the water binding regions of TSs can have profound effects on the hydroxylation activities exhibited by these enzymes. [78] …”