Rational Engineering of a Designed Protein Cage for siRNA Delivery

Oligonucleotide therapeutics have transformative potential in modern medicine but are poor drug candidates in themselves unless fitted with compensatory carrier systems. We describe a simple approach to transform a designed porous protein cage into a nucleic acid delivery vehicle. By introducing arginine mutations to the lumenal surface, a positively supercharged capsule is created, which can encapsidate oligonucleotides in vitro with high binding affinity. We demonstrate that the siRNA-loaded cage is taken up by mammalian cells and releases its cargo to induce RNAi and knockdown gene expression. These general concepts could also be applied to alternative scaffold designs, expediting the development of artificial protein cages toward delivery applications

Microwave-Assisted Solvent Extraction of Inert Platinum Group Metals from HNO₃(aq) to Betainium-Based Thermomorphic Ionic Liquid

Microwave irradiation efficiently assists extraction of substitution-inert platinum group metals like Ru­(III) and Rh­(III) from HNO₃(aq) to betainium bis­(trifluoromethylsulfonyl)­amide ([Hbet]­[Tf₂N]) ionic liquid to give nearly quantitative yields within 10¹–10² s. The rapid microwave heating also facilitates to mix the aqueous/IL biphasic system homogeneously without vigorous shaking because of its thermomorphic behavior. Rapid and efficient extraction of substitution-inert platinum group metals has been achieved in use of thermomorphic ionic liquid and microwave irradiation

Significant Acceleration of PGMs Extraction with UCST-Type Thermomorphic Ionic Liquid at Elevated Temperature

Dependency of extraction behavior of inert platinum group metals (PGMs) like Ru­(III) and Rh­(III) on temperature has been investigated in a biphasic system consisting of HNO₃(aq) and betainium bis­(trifluoromethylsulfonyl)­amide ([Hbet]­[Tf₂N]) ionic liquid. The extraction reactions of Ru­(III) and Rh­(III) took 3.5 days and 113 days, respectively, at 298 K, while equilibrated within 1 and 3 h to reach 99.2% and 96.5% extraction at 353 K. Further mechanistic studies clarified that the complexation of these PGMs and [Hbet]⁺ is rate-determining in their extraction and that it is successfully accelerated and enhanced by elevating the temperature

Complete Biosynthetic Pathway of Anditomin: Nature’s Sophisticated Synthetic Route to a Complex Fungal Meroterpenoid

Anditomin and its precursors, andilesins, are fungal meroterpenoids isolated from <i>Aspergillus variecolor</i> and have unique, highly oxygenated chemical structures with a complex bridged-ring system. Previous isotope-feeding studies revealed their origins as 3,5-dimethylorsellinic acid and farnesyl pyrophosphate and suggested the possible involvement of a Diels–Alder reaction to afford the congested bicyclo[2.2.2]­octane core structure of andilesins. Here we report the first identification of the biosynthetic gene cluster of anditomin and the determination of the complete biosynthetic pathway by characterizing the functions of 12 dedicated enzymes. The anditomin pathway actually does not employ a Diels–Alder reaction, but involves the nonheme iron-dependent dioxygenase AndA to synthesize the bridged-ring by an unprecedented skeletal reconstruction. Another dioxygenase, AndF, is also responsible for the structural complexification, generating the end product anditomin by an oxidative rearrangement

Structural and Mechanistic Insights into the C–C Bond-Forming Rearrangement Reaction Catalyzed by Heterodimeric Hinokiresinol Synthase

Hinokiresinol synthase (HRS) from Asparagus officinalis consists of two subunits, α and β, and catalyzes an unusual decarboxylative rearrangement reaction of 4-coumaryl 4-coumarate to generate (Z)-hinokiresinol with complete stereoselectivity. Herein, we describe the mechanism of rearrangement catalysis and the role played by the heterodimeric HRS, through structural and computational analyses. Our results suggest that the HRS reaction is unlikely to proceed via the previously hypothesized Claisen rearrangement mechanism. Instead, we propose that the 4-coumaryl 4-coumarate substrate is first cleaved into coumarate and an extended p-quinone methide, which then recombine to generate a new C–C bond. These processes are facilitated by proton transfers mediated by the basic residues (α-Lys164, α-Arg169, β-Lys168, and β-Arg173) in the cavity at the heterodimer interface. The active site residues, α-Asp165, β-Asp169, β-Trp17, β-Met136, and β-Ala171, play crucial roles in controlling the regioselectivity of the coupling between the fragmented intermediates as well as the stereoselectivity of the decarboxylation step, leading to the formation of the (Z)-hinokiresinol product

Prenylation of a Nonaromatic Carbon of Indolylbutenone by a Fungal Indole Prenyltransferase

FtmPT1 from <i>Aspergillus fumigatus</i> is a fungal indole prenyltransferase (PT) that normally catalyzes the regiospecific prenylation of brevianamide F (cyclo-l-Trp-l-Pro) at the C-2 position of the indole ring with dimethylallyl diphosphate (DMAPP). Interestingly, FtmPT1 exhibited remarkable substrate tolerance and accepted (<i>E</i>)-4-(1<i>H</i>-indol-3-yl)but-3-en-2-one (<b>1</b>) as a substrate to produce an unnatural novel α-prenylindolylbutenone (<b>1a</b>). This is the first demonstration of the prenylation of a nonaromatic carbon of the acceptor substrate by a fungal indole PT

Genetic Polymorphisms of Dihydropyrimidinase in a Japanese Patient with Capecitabine-Induced Toxicity

<div><p>Dihydropyrimidinase (DHP) is the second enzyme in the catabolic pathway of uracil, thymine, and chemotherapeutic fluoropyrimidine agents such as 5-fluorouracil (5-FU). Thus, DHP deficiency might be associated with 5-FU toxicity during fluoropyrimidine chemotherapy. We performed genetic analyses of the family of a patient with advanced colon cancer who underwent radical colectomy followed by treatment with 5-FU prodrug capecitabine and developed severe toxicity attributable to a lack of DHP. We measured urinary uracil and dihydrouracil, and genotyped <i>DPYS</i> in the patient and her family. We also measured the allele frequency of <i>DPYS</i> polymorphisms in 391 unrelated Japanese subjects. The patient had compound heterozygous missense and nonsense polymorphisms comprising c.1001A>G (p.Gln334Arg) in exon 6 and c.1393C>T (p.Arg465Ter) in exon 8, which are known to result in a DHP enzyme with little or no activity. The urinary dihydrouracil/uracil ratio in the patient was 17.08, while the mean ± SD urinary dihydrouracil/uracil ratio in family members who were heterozygous or homozygous for wild-type <i>DPYS</i> was 0.25 ± 0.06. In unrelated subjects, 8 of 391 individuals were heterozygous for the c.1001A>G mutation, while the c.1393C>T mutation was not identified. This is the first report of a DHP-deficient patient with <i>DPYS</i> compound heterozygous polymorphisms who was treated with a fluoropyrimidine, and our findings suggest that polymorphisms in the <i>DPYS</i> gene are pharmacogenomic markers associated with severe 5-FU toxicity in Japanese patients.</p></div

Catabolic pathway of 5-fluorouracil and uracil.

<p>5-FU and uracil are inactivated by dihydropyrimidine dehydrogenase to fluoro-5,6-dihydrouracil and 5,6-dihydrouracil, respectively, which are subsequently converted by dihydropyrimidinase to fluoro-β-ureidopropionate and β-ureidopropionate, respectively. Fluoro-β-alanine and β-alanine are the final catabolite in this cascade and are formed by β-ureidopropionase.</p

Discovery of Key Dioxygenases that Diverged the Paraherquonin and Acetoxydehydroaustin Pathways in <i>Penicillium brasilianum</i>

Paraherquonin (<b>1</b>), a fungal meroterpenoid produced by <i>Penicillium brasilianum</i> NBRC 6234, possesses a unique, highly congested hexacyclic molecular architecture. Here we identified the biosynthetic gene cluster of <b>1</b> (the <i>prh</i> cluster) and elucidated the pathway up to berkeleydione (<b>2</b>), which serves as the key intermediate for the biosynthesis of <b>1</b> as well as many other meroterpenoids. Interestingly, the nonheme iron and α-ketoglutarate-dependent dioxygenase PrhA constructs the cycloheptadiene moiety to afford <b>2</b> from preaustinoid A1 (<b>6</b>), probably via the homoallyl-homoallyl radical rearrangement. Additionally, another fungal strain, <i>P. brasilianum</i> MG11, which produces acetoxydehydroaustin instead of <b>1</b>, was found to have a gene cluster nearly identical to the <i>prh</i> cluster. The dioxygenase encoded by the cluster shares 92% sequence identity with PrhA, and also accepts <b>6</b> but produces preaustinoid A3 (<b>17</b>) with a spiro-lactone system, generating a diverging point for the two different meroterpenoid pathways in the same species

A Methyltransferase Initiates Terpene Cyclization in Teleocidin B Biosynthesis

Teleocidin B is an indole terpenoid isolated from Streptomyces. Due to its unique chemical structure and ability to activate protein kinase C, it has attracted interest in the areas of organic chemistry and cell biology. Here, we report the identification of genes encoding enzymes for teleocidin B biosynthesis, including nonribosomal peptide synthetase (<i>tleA</i>), P-450 monooxygenase (<i>tleB</i>), prenyltransferase (<i>tleC</i>), and methyltransferase (<i>tleD</i>). The <i>tleD</i> gene, which is located outside of the <i>tleABC</i> cluster on the chromosome, was identified by transcriptional analysis and heterologous expression. Remarkably, TleD not only installs a methyl group on the geranyl moiety of the precursor but also facilitates the nucleophilic attack from the electron-rich indole to the resultant cation, to form the indole-fused six-membered ring. This is the first demonstration of a cation, generated from methylation, triggering successive terpenoid ring closure