Reductive dehydroxylation of allyl alcohols by IspH protein

The biosynthesis of natural products is a treasure trove of unusual reaction mechanisms. This Minireview summarizes recent work on the structure and mechanism of IspH protein, which catalyzes the reductive dehydroxylation of an allyl alcohol in a biosynthetic pathway leading to isoprenoid precursors.     

Neuronal RNA granule contains ApCPEB1, a novel cytoplasmic polyadenylation element binding protein, in Aplysia sensory neuron

The cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) binds to CPE containing mRNAs on their 3'' untranslated regions (3''UTRs). This RNA binding protein comes out many important tasks, especially in learning and memory, by modifying the translational efficiency of target mRNAs via poly (A) tailing. Overexpressed CPEB has been reported to induce the formation of stress granules (SGs), a sort of RNA granule in mammalian cell lines. RNA granule is considered to be a potentially important factor in learning and memory. However, there is no study about RNA granule in Aplysia. To examine whether an Aplysia CPEB, ApCPEB1, forms RNA granules, we overexpressed ApCPEB1-EGFP in Aplysia sensory neurons. Consistent with the localization of mammalian CPEB, overexpressed ApCPEB1 formed granular structures, and was colocalized with RNAs and another RNA binding protein, ApCPEB, showing that ApCPEB1 positive granules are RNA-protein complexes. In addition, ApCPEB1 has a high turnover rate in RNA granules which were mobile structures. Thus, our results indicate that overexpressed ApCPEB1 is incorporated into RNA granule which is a dynamic structure in Aplysia sensory neuron. We propose that ApCPEB1 granule might modulate translation, as other RNA granules do, and furthermore, influence memory.     

Norcholanic acids as substrates for recombinant 3β-hydroxysteroid dehydrogenase and progesterone 5β-reductase, enzymes of the 5β-cardenolide biosynthesis

The conversion of 23-nor-5,20(22)E-choladienic acid-3β-ol and other intermediates of the putative norcholanic acid pathway of cardenolide biosynthesis by recombinant 3β-hydroxysteroid dehydrogenase from Digitalis lanata in dehydrogenation and reduction reactions was investigated. 23-nor-4,20(22)E-choladienic acid-3-one was found to be a substrate of recombinant progesterone 5β-reductases from D. lanata and Arabidopsis thaliana. The role of various substrates in cardenolide biosynthesis is discussed.     

Macrodiolide Formation by the Thioesterase of a Modular Polyketide Synthase

Elaiophylin is an unusual C₂‐symmetric antibiotic macrodiolide produced on a bacterial modular polyketide synthase assembly line. To probe the mechanism and selectivity of diolide formation, we sought to reconstitute ring formation in vitro by using a non‐natural substrate. Incubation of recombinant elaiophylin thioesterase/cyclase with a synthetic pentaketide analogue of the presumed monomeric polyketide precursor of elaiophylin, specifically its N‐acetylcysteamine thioester, produced a novel 16‐membered C₂‐symmetric macrodiolide. A linear dimeric thioester is an intermediate in ring formation, which indicates iterative use of the thioesterase active site in ligation and subsequent cyclization. Furthermore, the elaiophylin thioesterase acts on a mixture of pentaketide and tetraketide thioesters to give both the symmetric decaketide diolide and the novel asymmetric hybrid nonaketide diolide. Such thioesterases have potential as tools for the in vitro construction of novel diolides.     

Reconstitution of Biosynthetic Machinery for the Synthesis of the Highly Elaborated Indole Diterpene Penitrem

Penitrem A is one of the most elaborated members of the fungal indole diterpenes. Two separate penitrem gene clusters were identified using genomic and RNA sequencing data, and 13 out of 17 transformations in the penitrem biosynthesis were elucidated by heterologous reconstitution of the relevant genes. These reactions involve 1) a prenylation‐initiated cationic cyclization to install the bicyclo[3.2.0]heptane skeleton (PtmE), 2) a two‐step P450‐catalyzed oxidative processes forming the unique tricyclic penitrem skeleton (PtmK and PtmU), and 3) five sequential oxidative transformations (PtmKULNJ). Importantly, without conventional gene disruption, reconstitution of the biosynthetic machinery provided sufficient data to determine the pathway. It was thus demonstrated that the Aspergillus oryzae reconstitution system is a powerful method for studying the biosynthesis of complex natural products.     

Pinensins: The First Antifungal Lantibiotics

Lantibiotics (lanthionine‐containing antibiotics) from Gram‐positive bacteria typically exhibit activity against Gram‐positive bacteria. The activity and structure of pinensin A ( 1 ) and B ( 2 ), lantibiotics isolated from a native Gram‐negative producer Chitinophaga pinensis are described. Surprisingly, the pinensins were found to be highly active against many filamentous fungi and yeasts but show only weak antibacterial activity. To the best of our knowledge, lantibiotic fungicides have not been described before. An in‐depth bioinformatic analysis of the biosynthetic gene cluster established the ribosomal origin of these compounds and identified candidate genes encoding all of the enzymes required for post‐translational modification. Additional encoded functions enabled us to build up a hypothesis for the biosynthesis, export, sensing, and import of this intriguing lantibiotic.