Cyclization of Squalene from Both Termini: Identification of an Onoceroid Synthase and Enzymatic Synthesis of Ambrein

The onoceroids are triterpenoids biosynthesized from squalene or (3<i>S</i>)-2,3-oxidosqualene by cyclization from both termini. We recently revealed that tetraprenyl-β-curcumene cyclase from <i>Bacillus megaterium</i> (BmeTC) is a bifunctional triterpene/sesquarterpene cyclase that converts head-to-tail tetraprenyl-β-curcumene and tail-to-tail squalene into pentacyclic and bicyclic products, respectively, in vivo. Here, we reveal that BmeTC has an unprecedented catalytic function in cyclizing squalene from both termini and is the first onoceroid synthase. We also report the first onoceroids from bacterial origin. Our discoveries suggest that symmetric and asymmetric onoceroids could be biosynthesized by a single enzyme via an intermediate cyclized at one terminus of squalene. Furthermore, the new function of BmeTC enabled the synthesis of (+)-ambrein, a major constituent of ambergris that is difficult to obtain naturally, via a mutated squalene-hopene cyclase–catalyzed reaction from easily available squalene

Characterization of Class IB Terpene Synthase: The First Crystal Structure Bound with a Substrate Surrogate

Terpene synthases (TS) are classified into two broad types, Class I and II, based on the chemical strategy for initial carbocation formation and motif sequences of the catalytic site. We have recently identified a new class of enzymes, Class IB, showing the acceptability of long (C₂₀–C₃₅) prenyl-diphosphates as substrates and no amino acid sequence homology with known TS. Conversion of long prenyl-diphosphates such as heptaprenyl-diphosphate (C₃₅) is unusual and has never been reported for Class I and II enzymes. Therefore, the characterization of Class IB enzymes is crucial to understand the reaction mechanism of the extensive terpene synthesis. Here, we report the crystal structure bound with a substrate surrogate and biochemical analysis of a Class IB TS, using the enzyme from Bacillus alcalophilus (BalTS). The structure analysis revealed that the diphosphate part of the substrate is located around the two characteristic Asp-rich motifs, and the hydrophobic tail is accommodated in a unique hydrophobic long tunnel, where the C₃₅ prenyl-diphosphate, the longest substrate of BalTS, can be accepted. Biochemical analyses of BalTS showed that the enzymatic property, such as Mg^2⁺ dependency, is similar to those of Class I enzymes. In addition, a new cyclic terpene was identified from BalTS reaction products. Mutational analysis revealed that five of the six Asp residues in the Asp-rich motifs and two His residues are essential for the formation of the cyclic skeleton. These results provided a clue to consider the application of the unusual large terpene synthesis by Class IB enzymes

Bioinspired synthesis of pentacyclic onocerane triterpenoids

The first chemical synthesis of pentacyclic onocerane triterpenoids has been achieved. A putative biomimetic tricyclization cascade is employed to forge a fused decalin-/oxepane ring system. The synthetic route proceeds to (+)-cupacinoxepin in seven steps and to (+)-onoceranoxide in eight steps in the longest linear sequence, when starting from geranyl chloride and (+)-sclareolide. The bioinspired epoxypolyene cyclization is supported by computational and enzymatic studies

Bioinspired synthesis of pentacyclic onocerane triterpenoids.

The first chemical synthesis of pentacyclic onocerane triterpenoids has been achieved. A putative biomimetic tricyclization cascade is employed to forge a fused decalin-/oxepane ring system. The synthetic route proceeds to (+)-cupacinoxepin in seven steps and to (+)-onoceranoxide in eight steps in the longest linear sequence, when starting from geranyl chloride and (+)-sclareolide. The bioinspired epoxypolyene cyclization is supported by computational and enzymatic studies

Crystal structure and functional analysis of large-terpene synthases belonging to a newly found subclass

Thousands of terpenes have been identified to date. However, only two classes of enzymes are known to be involved in their biosynthesis, and each class has characteristic amino-acid motifs. We recently identified a novel large-terpene (C25/C30/C35) synthase, which shares no motifs with known enzymes. To elucidate the molecular mechanism of this enzyme, we determined the crystal structure of a large-β-prene synthase from B. alcalophilus (BalTS). Surprisingly, the overall structure of BalTS is similar to that of the α-domain of class I terpene synthases although their primary structures are totally different from each other. Two novel aspartate-rich motifs, DYLDNLxD and DY(F, L, W)IDxxED, are identified, and mutations of any one of the aspartates eliminate its enzymatic activity. The present work leads us to propose a new subclass of terpene synthases, class IB, which is probably responsible for large-terpene biosynthesis

Iatrogenic intramuscular hematoma of the oblique muscles as a complication of technetium-99m-labeled pyrophosphate imaging-based computed tomography-guided core-needle biopsy in a patient with wild-type transthyretin cardiac amyloidosis

Technetium-99m-labeled pyrophosphate imaging-based computed tomography-guided core-needle biopsy of the internal oblique muscle with tracer uptake is a safe and sensitive extracardiac screening biopsy. It can provide histopathological confirmation of the deposition of amyloid transthyretin in patients with wild-type transthyretin cardiac amyloidosis. This case report presents the case of a 73-year-old man receiving triple anti-thrombotic therapy for atrial flutter and coronary stenting who underwent this biopsy to confirm the diagnosis of transthyretin cardiac amyloidosis. The biopsy needle reached the internal oblique muscle via the external oblique muscle between the skin and the target. A type 1 intramuscular hematoma involving these muscles developed subsequently; however, manual compression hemostasis prevented further increase in size. Since this biopsy often targets elderly patients receiving anti-thrombotic therapy who are at high risk of bleeding owing to multimorbidity and polypharmacy, efforts should be made to reduce the frequency of complications, particularly bleeding, which can lead to the development of intramuscular hematoma

Le dispositif de VAE militante entre rescolarisation et transformations de l'engagement

La validation des acquis de l'expérience (VAE) est un dispositif public créé en 2002, qui permet aux individus d'obtenir des diplômes en faisant reconnaître par des institutions éducatives des activités et des pratiques comme équivalentes aux savoirs et aux savoir-faire acquis dans les formations qui mènent à ces diplômes. La VAE apparaît alors comme un élément de la transformation plus générale de la conception de la formation continue, en ce sens qu'elle met plutôt en scène une conception économiciste, individualiste et instrumentale de la formation, et tout particulièrement parce qu'elle s'appuie sur les deux " outils " socio-cognitifs qui en sont aujourd'hui le cœur : les compétences et le projet. L'étude de la mise en valeur dans les dossiers de VAE des expériences d'engagement montre de quelle manière ce dispositif contribue à développer une conception du militantisme en termes de compétences, en même temps qu'il permet d'insister sur les logiques de rattrapage scolaire qu'il induit