Magnesium-specific ring expansion/contraction catalysed by the class II diterpene cyclase from pleuromutilin biosynthesis.
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ABSTRACT:
SUBMITTER: Lemke C
PROVIDER: S-EPMC7430159 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Biosynthesis of the complex diterpenoid antibiotic pleuromutilin relies on a bifunctional (di)terpene synthase, and here site-directed mutagenesis was used to knockout either of the two active sites. This enabled characterization of the novel ring contracted intermediate produced by the initiating class II diterpene cyclase active site. Quantum chemical calculations further indicate the importance of reactant configuration for this intriguing ring rearrangement.
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Project description: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.
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Project description:Mycobacterium tuberculosis remains a widespread and devastating human pathogen, whose ability to infiltrate macrophage host cells from the human immune system is an active area of investigation. We have recently reported the discovery of a novel diterpene from M. tuberculosis, edaxadiene, whose ability to arrest phagosomal maturation in isolation presumably contributes to this critical process in M. tuberculosis infections. (Mann, F. M., Xu, M., Chen, X., Fulton, D. B., Russell, D. G., and Peters, R. J. (2009) J. Am. Chem. Soc., in press). Here, we present characterization of the class II diterpene cyclase that catalyzes the committed step in edaxadiene biosynthesis, i.e. the previously identified halimadienyl-diphosphate synthase (HPS; EC 5.5.1.16). Intriguingly, our kinetic analysis suggests a potential biochemical regulatory mechanism that triggers edaxadiene production upon phagosomal engulfment. Furthermore, we report characterization of potential HPS inhibitors: specifically, two related transition state analogs (15-aza-14,15-dihydrogeranylgeranyl diphosphate (7a) and 15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (7b)) that exhibit very tight binding. Although arguably not suitable for clinical use, these nevertheless provide a basis for pharmaceutical design against this intriguing biosynthetic pathway. Finally, we provide evidence indicating that this pathway exists only in M. tuberculosis and is not functional in the closely related Mycobacterium bovis because of an inactivating frameshift in the HPS-encoding gene. Thus, we hypothesize that the inability to produce edaxadiene may be a contributing factor in the decreased infectivity and/or virulence of M. bovis relative to M. tuberculosis in humans.
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Project description:A β-diketiminato n-butylmagnesium complex is presented as a selective precatalyst for the reductive hydroboration of organic nitriles with pinacolborane (HBpin). Stoichiometric reactivity studies indicate that catalytic turnover ensues through the generation of magnesium aldimido, aldimidoborate and borylamido intermediates, which are formed in a sequence of intramolecular nitrile insertion and inter- and intramolecular B-H metathesis events. Kinetic studies highlight variations in mechanism for the catalytic dihydroboration of alkyl nitriles, aryl nitriles bearing electron withdrawing (Ar(EWG)CN) and aryl nitriles bearing electron donating (Ar(EDG)CN) substitution patterns. Kinetic isotope effects (KIEs) for catalysis performed with DBpin indicate that B-H bond breaking and C-H bond forming reactions are involved in the rate determining processes during the dihydroboration of alkyl nitriles and Ar(EDG)CN substrates, which display divergent first and second order rate dependences on [HBpin] respectively. In contrast, the hydroboration of Ar(EWG)CN substrates provides no KIE and HBpin is not implicated in the rate determining process during catalysis. Irrespective of these differences, a common mechanism is proposed in which the rate determining steps are deduced to vary through the establishment of several pre-equilibria, the relative positions of which are determined by the respective stabilities of the dimeric and monomeric magnesium aldimide and magnesium aldimidoborate intermediates as a result of adjustments to the basicity of the nitrile substrate. More generally, these observations indicate that homogeneous processes performed under heavier alkaline earth catalysis are likely to demonstrate previously unappreciated mechanistic diversity.
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Project description:Terpene synthases are capable of mediating complex reactions, but fundamentally simply catalyze lysis of allylic diphosphate esters with subsequent deprotonation. Even with the initially generated tertiary carbocation this offers a variety of product outcomes, and deprotonation further can be preceded by the addition of water. This is particularly evident with labdane-related diterpenes (LRDs) where such lysis follows bicyclization catalyzed by class II diterpene cyclases (DTCs) that generates preceding structural variation. Previous investigation revealed that two diterpene synthases (DTSs), one bacterial and the other plant-derived, exhibit extreme substrate promiscuity, but yet still typically produce exo-ene or tertiary alcohol LRD derivatives, respectively (i.e., demonstrating high catalytic specificity), enabling rational combinatorial biosynthesis. Here two DTSs that produce either cis or trans endo-ene LRD derivatives, also plant and bacterial (respectively), were examined for their potential analogous utility. Only the bacterial trans-endo-ene forming DTS was found to exhibit significant substrate promiscuity (with moderate catalytic specificity). This further led to investigation of the basis for substrate promiscuity, which was found to be more closely correlated with phylogenetic origin than reaction complexity. Specifically, bacterial DTSs exhibited significantly more substrate promiscuity than those from plants, presumably reflecting their distinct evolutionary context. In particular, plants typically have heavily elaborated LRD metabolism, in contrast to the rarity of such natural products in bacteria, and the lack of potential substrates presumably alleviates selective pressure against such promiscuity. Regardless of such speculation, this work provides novel biosynthetic access to almost 19 LRDs, demonstrating the power of the combinatorial approach taken here.
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