Structure of a Three-Domain Sesquiterpene Synthase: A Prospective Target for Advanced Biofuels Production

Abstract

The sesquiterpene bisabolene was recently identified as a biosynthetic precursor to bisabolane, an advanced biofuel with physicochemical properties similar to those of D2 diesel. High-titer microbial bisabolene production was achieved using Abies grandis α-bisabolene synthase (AgBIS). Here, we report the structure of AgBIS, a three-domain plant sesquiterpene synthase, crystallized in its apo form and bound to five different inhibitors. Structural and biochemical characterization of the AgBIS terpene synthase Class I active site leads us to propose a catalytic mechanism for the cyclization of farnesyl diphosphate into bisabolene via a bisabolyl cation intermediate. Further, we describe the nonfunctional AgBIS Class II active site whose high similarity to bifunctional diterpene synthases makes it an important link in understanding terpene synthase evolution. Practically, the AgBIS crystal structure is important in future protein engineering efforts to increase the microbial production of bisabolene.

Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase

“…The discovery of golden larch PxaTPS8, which forms a 5,7-fused bicyclic diterpene, here coined pseudolaratriene, adds a hitherto unique catalyst to the diTPS family with possible uses for PAB biomanufacture. The concordant occurrence of PxaTPS8, its product pseudolaratriene, and PAB in P. amabilis root, bark, and needle tissue supports the intermediacy of pseudolaratriene in (19) showing the typical diTPS three-domain structure including the γ-, β-, and α-domains (magenta, cyan, and blue, respectively). (Left) Stereoview of the PxaTPS8 active site with docked GGPP (yellow), DDxxD motif (red), and NSE/DTE motif (gray).…”

“…The four BIS-like candidates identified in the golden larch root transcriptome resembled three-domain class I TPSs, lacking a class II active site and featuring the DDxxD and Asn-Ser-Glu/Asp-Thr-Glu (NSE/DTE) class I catalytic motifs (19). A γβα-domain structure is conserved among enzymes of the TPS-d3 clade.…”

“…We conducted homology modeling of PxaTPS8 based on the structure of grand fir α-BIS (AgBIS) (19) and molecular docking of GGPP into the active site to probe residues that contribute to the distinct activity of PxaTPS8 (Fig. 4).…”

“…4). Among common features, the Mg 2+ -coordinating DDxxD motif and three arginines (R558, R560, and R736) shown to be critical for catalysis in AgBIS (19) and other class I TPSs (26) are conserved in PxaTPS8. Concurrently, alanine substitution of R558 as a representative of the catalytic arginine triad abolished activity in E. coli in vivo assays (Fig.…”

“…Within an 8-Å radius around the docked ligand in the PxaTPS8 active site cavity, several residues with possible catalytic impact were identified, including positions known to direct product outcome in class I TPSs, such as the hinge region on helix G (Fig. 4) (19,26,27). Substitution of PxaTPS8 S696, G697, and A701 for different functional residues common in other gymnosperm TPSs led to a decrease (S696I/V, G697S, and A701C) or loss (A701L) of pseudolaratriene formation.…”