Two bacterial diterpene synthases from the actinomycete Allokutzneria albata were investigated, resulting in the identification of the structurally unprecedented compound spiroalbatene from the first ...and cembrene A from the second enzyme. Both enzymes were thoroughly investigated in terms of their mechanisms by isotope labeling experiments, site‐directed mutagenesis, and variation of the metal cofactors and pH value. For spiroalbatene synthase, the pH‐ and Mn2+‐dependent formation of the side product thunbergol was observed, which is biosynthetically linked to spiroalbatene.
Two diterpene synthases from Allokutzneria albata were investigated in terms of their products, which resulted in the identification of the structurally complex compound spiroalbatene and cembrene A. The enzyme mechanisms were studied by isotope labeling experiments, through mutations, and by variation of the incubation conditions. For spiroalbatene synthase, a change in the incubation conditions resulted in the formation of thunbergol.
Small-ring cage hydrocarbons are popular bioisosteres (molecular replacements) for commonly found para-substituted benzene rings in drug design1. The utility of these cage structures derives from ...their superior pharmacokinetic properties compared with their parent aromatics, including improved solubility and reduced susceptibility to metabolism2,3. A prime example is the bicyclo1.1.1pentane motif, which is mainly synthesized by ring-opening ofthe interbridgehead bond of the strained hydrocarbon 1.1.1propellane with radicals or anions4. By contrast, scaffolds mimicking meta-substituted arenes are lacking because of the challenge of synthesizing saturated isosteres that accurately reproduce substituent vectors5. Here we show that bicyclo3.1.1heptanes (BCHeps), which are hydrocarbons for which the bridgehead substituents map precisely onto the geometry of meta-substituted benzenes, can be conveniently accessed from 3.1.1propellane. We found that 3.1.1 propellane can be synthesized on a multigram scale, and readily undergoes a range of radical-based transformations to generate medicinally relevant carbon- and heteroatom-substituted BCHeps, including pharmaceutical analogues. Comparison of the absorption, distribution, metabolism and excretion (ADME) properties of these analogues reveals enhanced metabolic stability relative to their parent arene-containing drugs, validating the potential of this meta-arene analogue as an sp3-rich motif in drug design. Collectively, our results show that BCHeps can be prepared on useful scales using a variety of methods, offering a new surrogate for mefa-substituted benzene rings for implementation in drug discovery programmes.
We present crystallographic and functional data of selina‐4(15),7(11)‐diene synthase (SdS) from Streptomyces pristinaespiralis in its open and closed (ligand‐bound) conformation. We could identify an ...induced‐fit mechanism by elucidating a rearrangement of the G1/2 helix‐break motif upon substrate binding. This rearrangement highlights a novel effector triad comprising the pyrophosphate sensor Arg178, the linker Asp181, and the effector Gly182‐O. This structural motif is strictly conserved in class I terpene cyclases from bacteria, fungi, and plants, including epi‐isozizaene synthase (3KB9), aristolochene synthase (4KUX), bornyl diphosphate synthase (1N20), limonene synthase (2ONG), 5‐epi‐aristolochene synthase (5EAT), and taxa‐4(5),11(12)‐diene synthase (3P5R). An elaborate structure‐based mutagenesis in combination with analysis of the distinct product spectra confirmed the mechanistic models of carbocation formation and stabilization in SdS.
A class act: The open and closed (ligand‐bound) conformations of selinadiene synthase reveal an induced‐fit mechanism, and give insights into carbocation formation and regulation in class I terpene cyclases. The substrate turnover is accomplished by an effector triad on the G1 helix, comprising the pyrophosphate sensor Arg178, the linker Asp181, and the effector Gly182. This structural motif is strictly conserved in all class I terpene cyclases.
Stereospecifically labelled precursors were subjected to conversion by seven bacterial sesquiterpene cyclases to investigate the stereochemistry of their initial 1,10‐cyclisation‐1,3‐hydride shift ...cascades. Enzymes with products of known absolute configuration showed a coherent stereochemical course, except for (−)‐α‐amorphene synthase, for which the obtained results are better explained by an initial 1,6‐cyclisation. The link between the absolute configuration of the product and the stereochemical course of the 1,3‐hydride shifts enabled assignment of the absolute configurations of three enzyme products, which were confirmed independently through the absolute configuration of the common byproduct germacrene D‐4‐ol.
Jump ahead: Incubation experiments with (11‐13C,1,1‐2H2)FPP and stereospecifically labelled (1R)‐ and (1S)‐(1‐2H)FPP enabled assignment of the previously unknown absolute configurations of three bacterial sesquiterpenes from 1,3‐hydride shifts, and provided first experimental evidence for a cyclization mechanism to amorphanes that was previously proposed on the basis of quantum chemical calculations.
The mechanisms of two diterpene cyclases from streptomycetes—one with an unknown product that was identified as the spirocyclic hydrocarbon spiroviolene and one with the known product ...tsukubadiene—were investigated in detail by isotope labeling experiments. Although the structures of the products were very different, the cyclization mechanisms of both enzymes proceed through the same initial cyclization reactions, before they diverge towards the individual products, which is reflected in the close phylogenetic relationship of the enzymes.
A close relationship: The products of two phylogenetically related diterpene cyclases from streptomycetes were identified. The mechanisms of the enzymes were investigated by extensive labeling experiments. Although the structures of the diterpenes are very different, some steps of the cyclization cascade are very similar, thus suggesting that only minor conformational changes of the substrate are responsible for the formation of the unique products.
Terpene Cyclases from Social Amoebae Rabe, Patrick; Rinkel, Jan; Nubbemeyer, Britta ...
Angewandte Chemie (International ed.),
December 5, 2016, Letnik:
55, Številka:
49
Journal Article
Recenzirano
Genome sequences of social amoebae reveal the presence of terpene cyclases (TCs) in these organisms. Two TCs from Dictyostelium discoideum converted farnesyl diphosphate into ...(2S,3R,6S,9S)‐(−)‐protoillud‐7‐ene and (3S)‐(+)‐asterisca‐2(9),6‐diene. The enzyme mechanisms and EI‐MS fragmentations of the products were studied by labeling experiments.
Terpene time: The products of two terpene cyclases from the social amoeba Dictyostelium discoideum were identified as (2S,3R,6S,9S)‐protoillud‐7‐ene and (3S)‐asterisca‐2(9),6‐diene. The enzyme mechanisms and the EI‐MS fragmentation reactions of their products were studied in extensive isotopic labeling experiments.
Terpenes are structurally diverse natural products involved in many ecological interactions. The pivotal enzymes for terpene biosynthesis, terpene synthases (TPSs), had been described only in plants ...and fungi in the eukaryotic domain. In this report, we systematically analyzed the genome sequences of a broad range of nonplant/nonfungus eukaryotes and identified putative TPS genes in six species of amoebae, five of which are multicellular social amoebae from the order of Dictyosteliida. A phylogenetic analysis revealed that amoebal TPSs are evolutionarily more closely related to fungal TPSs than to bacterial TPSs. The social amoeba Dictyostelium discoideum was selected for functional study of the identified TPSs. D. discoideum grows as a unicellular organism when food is abundant and switches from vegetative growth to multicellular development upon starvation. We found that expression of most D. discoideum TPS genes was induced during development. Upon heterologous expression, all nine TPSs from D. discoideum showed sesquiterpene synthase activities. Some also exhibited monoterpene and/or diterpene synthase activities. Direct measurement of volatile terpenes in cultures of D. discoideum revealed essentially no emission at an early stage of development. In contrast, a bouquet of terpenes, dominated by sesquiterpenes including β-barbatene and (E,E)-α-farnesene, was detected at the middle and late stages of development, suggesting a development-specific function of volatile terpenes in D. discoideum. The patchy distribution of TPS genes in the eukaryotic domain and the evidence for TPS function in D. discoideum indicate that the TPS genes mediate lineage-specific adaptations.
The products obtained by incubation of farnesyl diphosphate (FPP) with six purified bacterial terpene cyclases were characterised by one- and two-dimensional NMR spectroscopic methods, allowing for a ...full structure elucidation. The absolute configurations of four terpenes were determined based on their optical rotary powers. Incubation experiments with
C-labelled isotopomers of FPP in buffers containing water or deuterium oxide allowed for detailed insights into the cyclisation mechanisms of the bacterial terpene cyclases.
Ivosidenib, an inhibitor of isocitrate dehydrogenase 1 (IDH1) R132C and R132H variants, is approved for the treatment of acute myeloid leukaemia (AML). Resistance to ivosidenib due to a second site ...mutation of IDH1 R132C, leading to IDH1 R132C/S280F, has emerged. We describe biochemical, crystallographic, and cellular studies on the IDH1 R132C/S280F and R132H/S280F variants that inform on the mechanism of second-site resistance, which involves both modulation of inhibitor binding at the IDH1 dimer-interface and alteration of kinetic properties, which enable more efficient 2-HG production relative to IDH1 R132C and IDH1 R132H. Importantly, the biochemical and cellular results demonstrate that it should be possible to overcome S280F mediated resistance in AML patients by using alternative inhibitors, including some presently in phase 2 clinical trials.
Dimethylsulfoniopropionate is a highly abundant sulfur metabolite in marine ecosystems. Its biosynthesis by different organisms including plants, marine algae and dinoflagellates is discussed. ...Furthermore, the accumulated knowledge about bacterial uptake systems and its climatically relevant degradation by marine bacteria to methanethiol or dimethylsulfide is presented. Finally, uptake and degradation of synthetic DMSP analogs are addressed.
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