Language：English   Publishing type：Research paper (scientific journal)  

Antihypercholesterolemic agent phomoidride (PMD) B has a highly elaborated bicyclo[4.3.1]deca-1,6-diene core scaffold derived from dimeric anhydride with a nine-membered ring. This report elucidated the late stage transformation from an anhydride monomer to PMD B through the heterologous expression of three enzyme genes, TstC, TstK, and TstE. Additional in vitro studies of TstK and TstE provided evidence on the formation of PMD via dimerization, three-step oxidation, and unusual methylation-triggered bicyclic ketal formation. Elucidation of the function of cyclase TstC prompts us to examine the cyclization mechanism of TstC by using a computational approach. Computational analytical data on PMD and structurally related glaucanic acid indicated that the initial decarboxylation of monomer results in enolate and subsequent double Michael reactions of another monomer, followed by an optional aldol reaction proceeding in an endo-selective manner to give cycloadducts, supporting the fact that the starting orientation of two monomers is directly transferred to the product configurations.

DOI： 10.1021/jacs.2c09308

PubMed

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acs.joc.2c00291

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

DOI： 10.1021/jacs.1c08336

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Pharmaceutical Society of Japan  

DOI： 10.1248/cpb.c21-00536

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

DOI： 10.1021/jacsau.1c00178

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society  

Brasilane-type sesquiterpenes have been known for a long time, but their biosynthetic pathways and mechanisms remain elusive. Recently, two groups independently characterized a Trichoderma terpene cyclase that produces trichobrasilenol, a brasilane-type sesquiterpene, and a plausible biosynthetic pathway was proposed based on isotopic labeling experiments. In the proposed mechanism, the characteristic brasilane-type 5/6 bicyclic skeleton is synthesized from a 5/7/3 tricyclic intermediate via a complicated concerted reaction, including six chemical events of C-C σ bond metathesis and rearrangements, ring-contraction, π bond formation, and regioselective hydroxylation. However, our density functional theory (DFT) calculations do not support this mechanism. On the basis of DFT calculations, we propose a new pathway for trichobrasilenol biosynthesis, involving a multistep carbocation cascade in which cyclopropylcarbinyl cations in equilibrium with homoallyl cations play a pivotal role. This pathway and mechanism is in good agreement with previous biosynthetic studies on brasilane-type compounds and related terpenoids, including isotope-labeling experiments and byproducts analysis.

DOI： 10.1021/jacs.0c09616

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Lolitrems are tremorgenic indole diterpenes that exhibit a unique 5/6 bicyclic system of the indole moiety. Although genetic analysis has indicated that the prenyltransferase LtmE and the cytochrome P450 LtmJ are involved in the construction of this unique structure, the detailed mechanism remains to be elucidated. Herein, we report the reconstitution of the biosynthetic pathway for lolitrems employing a recently established genome-editing technique for the expression host Aspergillus oryzae. Heterologous expression and bioconversion of the various intermediates revealed that LtmJ catalyzes multistep oxidation to furnish the lolitrem core. We also isolated the key reaction intermediate with an epoxyalcohol moiety. This observation allowed us to establish the mechanism of radical-induced cyclization, which was firmly supported by density functional theory calculations and a model experiment with a synthetic analogue.

DOI： 10.1002/anie.202007280

PubMed

Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/anie.202007280

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Pharmaceutical Society of Japan  

Preasperterpenoid A, featuring a 5/7/(3)6/5 pentacyclic structure, is a C25 sesterterpenoid produced by Penicillium verruculosum. The results of density functional calculations on putative biosynthetic carbocation cyclization/rearrangements leading to preasperterpenoid A revealed a highly concerted four-step cyclization mechanism. Interestingly, two secondary carbocation structures were obtained as minima, but appeared almost as shoulders in the energy profile, and may represent essentially transient structures during the highly concerted reaction.

DOI： 10.1248/cpb.c20-00037

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Shimalactones A and B are neuritogenic polyketides possessing characteristic oxabicyclo[2.2.1]heptane and bicyclo[4.2.0]octadiene ring systems that are produced by the marine fungus Emericella variecolor GF10. We identified a candidate biosynthetic gene cluster and conducted heterologous expression analysis. Expression of ShmA polyketide synthase in Aspergillus oryzae resulted in the production of preshimalactone. Aspergillus oryzae and Saccharomyces cerevisiae transformants expressing ShmA and ShmB produced shimalactones A and B, thus suggesting that the double bicyclo-ring formation reactions proceed non-enzymatically from preshimalactone epoxide. DFT calculations strongly support the idea that oxabicyclo-ring formation and 8π-6π electrocyclization proceed spontaneously after opening of the preshimalactone epoxide ring through protonation. We confirmed the formation of preshimalactone epoxide in vitro, followed by its non-enzymatic conversion to shimalactones in the dark.

DOI： 10.1002/anie.202001024

PubMed

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

We previously showed that the regio- and stereoselectivity in terpene-forming reactions are determined by the conformations of the carbocation intermediates, which reflect the initial conformation of the substrate, geranylfarnesyl diphosphate (GFPP). However, it remains unclear how the initial conformation of GFPP is controlled, and which part(s) of the GFPP molecule are important for its fixation inside the substrate-binding pocket. Here, we present the first detailed analysis of the inherent atomic mobility in carbocation intermediates during sesterterpene biosynthesis. We identified two methyl groups as the least mobile of all the carbons of the carbocation intermediates in the first half of the cyclization cascade. Our analysis suggests that these two methyl groups are critical for the preorganization of GFPP in the biosynthetic pathways leading to sesterfisherol and quiannulatene.

DOI： 10.3762/bjoc.15.184

PubMed

Authorship：Lead author   Language：Japanese   Publishing type：(MISC) Introduction and explanation (scientific journal)  

DOI： 10.14894/faruawpsj.55.7_684

Authorship：Lead author   Language：English   Publishing type：(MISC) Introduction and explanation (scientific journal)  

This review describes the application of computational chemistry to plant secondary metabolism, focusing on the biosynthetic mechanisms of terpene/terpenoid, alkaloid, flavonoid, and lignin as representative examples. Through these biosynthetic studies, we exhibit several computational methods, including density functional theory (DFT) calculations, theozyme calculation, docking simulation, molecular dynamics (MD) simulation, and quantum mechanics/molecular mechanics (QM/MM) calculation. This review demonstrates how modern computational chemistry can be employed as an effective tool for revealing biosynthetic mechanisms and the potential of computational chemistry-for example, elucidating how enzymes regulate regio- and stereoselectivity, finding the key catalytic residue of an enzyme, and assessing the viability of hypothetical pathways. Furthermore, insights for the next research objective involving application of computational chemistry to plant secondary metabolism are provided herein. This review will be helpful for plant scientists who are not well versed with computational chemistry.

DOI： 10.3389/fpls.2019.00802

PubMed

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The results of quantum chemical calculations on the mechanism of the carbocation cascade of reactions in the biosynthetic pathways leading to the pentacyclic sesterterpenes quiannulatene and sesterfisherol provide reasonable answers to several persistent mechanistic questions in sesterterpene biosynthesis, including: 1) the reaction pathways of the multicyclic ring system construction and skeletal rearrangements, 2) the mechanism of triquinane skeleton formation, which requires more complicated rearrangements than previously proposed, 3) the stereochemistry of the final carbocation intermediate, and 4) the determining factor of biosynthetic selection for either 5/6/4/6/5 or 5/6/5/5/5 pentacyclic skeleton formation. This in-depth mechanistic study on sesterterpene biosynthesis revealed that the shape of the final product and the type of triquinane skeleton formed are regulated by the stereochemistry and conformation of the common starting material, geranylfarnesyl diphosphate (GFPP).

DOI： 10.1002/anie.201807139

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society  

AndA, an Fe(II)/α-ketoglutarate (αKG)-dependent enzyme, is the key enzyme that constructs the unique and congested bridged-ring system of anditomin (1), by catalyzing consecutive dehydrogenation and isomerization reactions. Although we previously characterized AndA to some extent, the means by which the enzyme facilitates this drastic structural reconstruction have remained elusive. In this study, we have solved three X-ray crystal structures of AndA, in its apo form and in the complexes with Fe(II), αKG, and two substrates. The crystal structures and mutational experiments identified several key amino acid residues important for the catalysis and provided insight into how AndA controls the reaction. Furthermore, computational calculations validated the proposed reaction mechanism for the bridged-ring formation and also revealed the requirement of a series of conformational changes during the transformation.

DOI： 10.1021/jacs.8b06084

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

In the late stage of anthocyanin biosynthesis, dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS) mediate a formal tautomerization. However, such oxidation/reduction process requires high energy and appears to be unnecessary, as the oxidation state does not change during the transformation. Thus, a non-enzymatic pathway of tautomerization has also been proposed. To resolve the long-standing issue of whether this non-enzymatic pathway is the main contributor for the biosynthesis, we carried out density functional theory (DFT) calculations to examine this non-enzymatic pathway from dihydroflavonol to anthocyanidin. We show here that the activation barriers for the proposed non-enzymatic tautomerization are too high to enable the reaction to proceed under normal aqueous conditions in plants. The calculations also explain the experimentally observed requirement for acidic conditions during the final step of conversion of 2-flaven-3,4-diol to anthocyanidin; a thermodynamically and kinetically favorable concerted pathway can operate under these conditions.

DOI： 10.1371/journal.pone.0198944

PubMed

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The cyclization mechanisms involved in the biosynthesis of sesterterpenes are not fully understood. For example, there are two plausible reaction pathways for sesterfisherol biosynthesis, which differ in the order of ring cyclization: A-D-B/C (Path a) and A-B-C/D (Path b). It is difficult to capture intermediates of terpene cyclization, which is a complex, domino-type reaction, and so here we employed a combination of experimental and computational methods. Density functional theory calculations revealed unexpected intermediates and transition states, and implied that C-H···π interaction between a carbocation intermediate and an aromatic residue of sesterfisherol synthase (NfSS) plays a critical role, serving to accelerate the 1,2-H shift (thereby preventing triquinane carbocation formation) and to protect reactive carbocation intermediates from bases such as pyrophosphate or water in the active site. Site-directed mutagenesis of NfSS guided by docking simulations confirmed that phenylalanine F191 is a critical amino acid residue for sesterfisherol synthase, as the F191A mutant of NfSS produces novel sesterterpenes, but not sesterfisherol. Although both pathways are energetically viable, on the basis of our computational and experimental results, NfSS-mediated sesterfisherol biosynthesis appears to proceed via Path a. These findings may also provide new insight into the cyclization mechanisms in related sesterterpene synthases.

DOI： 10.1038/s41598-018-20916-x

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Heterologous expression of four clade-A bifunctional terpene synthases (BFTSs), giving di/sesterterpenes with unique polycyclic carbon skeletons such as sesterfisherol, enabled the isolation of the sesterterpenes Bm1, Bm2, Bm3, and Pb1. Their structures suggested that formation of the products occurs via various diastereomeric carbocation intermediates, allowing the proposal that clade-A BFTSs catalyze three-step cyclizations using several stereofacial combinations of allylic cation-olefin pairs in the corresponding intermediates to generate various stereoisomers.

DOI： 10.1021/acs.orglett.7b03418

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Event date： 2024.3

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：日本大学  

Event date： 2023.10

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Venue：千葉大学  

Event date： 2023.10

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Venue：東京理科大学薬学部  

Event date： 2023.10

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Venue：伊豆山研修センター  

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Event date： 2023.6

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Venue：千葉工業大学  

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Language：English   Presentation type：Symposium workshop panel(nominated)  

Venue：Kyoto  

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Event date： 2023.3

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Event date： 2023.3

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Event date： 2023.3

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Venue：オンライン  

Event date： 2023.3

Language：Japanese   Presentation type：Oral presentation(general)  

Event date： 2022.11

Language：Japanese   Presentation type：Poster presentation  

Venue：千葉大学  

Event date： 2022.10

Language：Japanese   Presentation type：Symposium workshop panel(nominated)  

Venue：シミックプラザ（山梨大学医学部構内）１階　シミックホール  

Event date： 2022.10

Language：Japanese   Presentation type：Poster presentation  

Venue：東京  

Event date： 2022.10

Language：Japanese   Presentation type：Poster presentation  

Venue：東京  

Event date： 2021.11

Language：Japanese   Presentation type：Oral presentation(invited, special)  

Venue：名古屋  

Event date： 2021.8

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Venue：オンライン  

Event date： 2021.3

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Event date： 2021.3

Language：Japanese   Presentation type：Media report,etc.  

Event date： 2021.2

Language：Japanese   Presentation type：Media report,etc.