Abstract
Pimarane-type diterpenoids are widely distributed in all domains of life, but no structures or catalytic mechanisms of pimarane-type diterpene synthases (DTSs) have been characterized. Here, we report that two class I DTSs, Sat1646 and Stt4548, each accept copalyl diphosphate (CPP) as the substrate to produce isopimara-8,15-diene (1). Sat1646 can also accept syn-CPP and produce syn-isopimaradiene/pimaradiene analogues (2–7), among which 2 possesses a previously unreported "6/6/7" ring skeleton. We solve the crystal structures of Sat1646, Sat1646 complexed with magnesium ions, and Stt4548, thereby revealing the active sites of these pimarane-type DTSs. Substrate modeling and subsequent site-directed mutagenesis experiments demonstrate different structural bases of Sat1646 and Stt4548 for 1 production. Comparisons with previously reported DTSs reveal their distinct carbocation intermediate stabilization mechanisms, which control the conversion of a single substrate CPP into structurally diverse diterpene products. These results illustrate the structural bases for enzymatic catalyses of pimarane-type DTSs, potentially facilitating future DTS engineering and combinatorial biosynthesis.
Highlights
Pimarane-type diterpenoids are widely distributed in all domains of life, but no structures or catalytic mechanisms of pimarane-type diterpene synthases (DTSs) have been characterized
In contrast to the very large number of diterpenoids (~18,000) that have been chemically characterized to date[10], much less is known about the catalytic mechanisms through which DTSs generate this vast structural diversity: only 11 DTSs structures have been solved (Fig. S2), and no structures or catalytic mechanisms have been demonstrated for pimarane-type DTSs
Comparisons of Sat1646/Stt4548 with other structurally characterized DTSs showed that stabilization of distinct carbocation intermediates underlies the conversion of a single substrate (CPP) into diverse diterpene products
Summary
Pimarane-type diterpenoids are widely distributed in all domains of life, but no structures or catalytic mechanisms of pimarane-type diterpene synthases (DTSs) have been characterized. Comparisons with previously reported DTSs reveal their distinct carbocation intermediate stabilization mechanisms, which control the conversion of a single substrate CPP into structurally diverse diterpene products These results illustrate the structural bases for enzymatic catalyses of pimarane-type DTSs, potentially facilitating future DTS engineering and combinatorial biosynthesis. In contrast to the very large number of diterpenoids (~18,000) that have been chemically characterized to date[10], much less is known about the catalytic mechanisms through which DTSs generate this vast structural diversity: only 11 DTSs structures have been solved (Fig. S2), and no structures or catalytic mechanisms have been demonstrated for pimarane-type DTSs. we report the functional and structural characterization of two pimarane-type DTSs, Sat1646 and Stt4548, both of which catalyze the biosynthesis of isopimara-8,15-diene (1, a known compound) using normal-copalyl diphosphate (normalCPP, designated CPP in descriptions below) as the substrate. Comparisons of Sat1646/Stt4548 with other structurally characterized DTSs showed that stabilization of distinct carbocation intermediates underlies the conversion of a single substrate (CPP) into diverse diterpene products (isopimaradiene/pimaradiene, abietadiene or biformene). Our study demonstrates the catalytic mechanism for pimarane-type DTSs and sets the stage for DTS engineering and combinatorial biosynthesis to obtain highly diverse pimarane-type diterpenoids
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