“Head‐to‐Middle” and “Head‐to‐Tail” cis‐Prenyl Transferases: Structure of Isosesquilavandulyl Diphosphate Synthase

We report the first X‐ray crystallographic structure of the “head‐to‐middle” prenyltransferase, isosesquilavandulyl diphosphate synthase, involved in biosynthesis of the merochlorin class of antibiotics. The protein adopts the ζ or cis‐prenyl transferase fold but remarkably, unlike tuberculosinol adenosine synthase and other cis‐prenyl transferases (e.g. cis‐farnesyl, decaprenyl, undecaprenyl diphosphate synthases), the large, hydrophobic side chain does not occupy a central hydrophobic tunnel. Instead, it occupies a surface pocket oriented at 90° to the hydrophobic tunnel. Product chain‐length control is achieved by squeezing out the ligand from the conventional allylic S1 binding site, with proton abstraction being achieved using a diphosphate‐Asn‐Ser relay. The structures revise and unify our thinking as to the mechanism of action of many other prenyl transferases and may also be of use in engineering new merochlorin‐class antibiotics.     

Aplysiasecosterol A: A 9,11‐Secosteroid with an Unprecedented Tricyclic γ‐Diketone Structure from the Sea Hare Aplysia kurodai

A new 9,11‐secosteroid having an unprecedented tricyclic γ‐diketone structure, aplysiasecosterol A ( 1 ), was isolated from the sea hare Aplysia kurodai. The structure was determined by one‐ and two‐dimensional NMR spectroscopic analysis, molecular modeling studies, a comparison of experimental and calculated ECD spectra, and a modified Mosher′s method. Aplysiasecosterol A ( 1 ) exhibited cytotoxicity against human myelocytic leukemia HL‐60 cells. A biosynthetic pathway for 1 from a known cholesterol was proposed and includes twice α‐ketol rearrangements and an intramolecular acetalization.     

The Source of “Fairy Rings”: 2‐Azahypoxanthine and its Metabolite Found in a Novel Purine Metabolic Pathway in Plants

Rings or arcs of fungus‐stimulated plant growth occur worldwide; these are commonly referred to as “fairy rings”. In 2010, we discovered 2‐azahypoxanthine (AHX), a compound responsible for the fairy‐ring phenomenon caused by fungus; AHX stimulated the growth of all the plants tested. Herein, we reveal the isolation and structure determination of a common metabolite of AHX in plants, 2‐aza‐8‐oxohypoxanthine (AOH). AHX is chemically synthesized from 5‐aminoimidazole‐4‐carboxamide (AICA), and AHX can be converted into AOH by xanthine oxidase. AICA is one of the members of the purine metabolic pathway in animals, plants, and microorganisms. However, further metabolism of AICA remains elusive. Based on these results and facts, we hypothesized that plants themselves produce AHX and AOH through a pathway similar to the chemical synthesis. Herein, we demonstrate the existence of endogenous AHX and AOH and a novel purine pathway to produce them in plants.     

Pinpointing a Mechanistic Switch Between Ketoreduction and “Ene” Reduction in Short‐Chain Dehydrogenases/Reductases

Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (?)‐menthone:(?)‐menthol reductase and (?)‐menthone:(+)‐neomenthol reductase, and the “ene” reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue‐swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β‐unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases.     

Structures of Iridoid Synthase from Cantharanthus roseus with Bound NAD+, NADPH,or NAD+/10‐Oxogeranial: Reaction Mechanisms

Structures of the iridoid synthase nepetalactol synthase in the presence of NAD⁺, NADPH or NAD⁺/10‐oxogeranial were solved. The 10‐oxogeranial substrate binds in a transoid‐O1‐C3 conformation and can be reduced by hydride addition to form the byproduct S‐10‐oxo‐citronellal. Tyr178 Oζ is positioned 2.5 Å from the substrate O1 and provides the second proton required for reaction. Nepetalactol product formation requires rotation about C1–C2 to form the cisoid isomer, leading to formation of the cis‐enolate, together with rotation about C4–C5, which enables cyclization and lactol production. The structure is similar to that of progesterone‐5β‐reductase, with almost identical positioning of NADP, Lys146(147), Tyr178(179), and F342(343), but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10‐oxogeranial structure also serves as a model for β‐face hydride attack in progesterone 5β‐reductases and is of general interest in the context of asymmetric synthesis.     

Injury‐Triggered Blueing Reactions of Psilocybe “Magic” Mushrooms

Upon injury, psychotropic psilocybin‐producing mushrooms instantly develop an intense blue color, the chemical basis and mode of formation of which has remained elusive. We report two enzymes from Psilocybe cubensis that carry out a two‐step cascade to prepare psilocybin for oxidative oligomerization that leads to blue products. The phosphatase PsiP removes the 4‐O‐phosphate group to yield psilocin, while PsiL oxidizes its 4‐hydroxy group. The PsiL reaction was monitored by in situ ¹³C NMR spectroscopy, which indicated that oxidative coupling of psilocyl residues occurs primarily via C‐5. MS and IR spectroscopy indicated the formation of a heterogeneous mixture of preferentially psilocyl 3‐ to 13‐mers and suggest multiple oligomerization routes, depending on oxidative power and substrate concentration. The results also imply that phosphate ester of psilocybin serves a reversible protective function.     

Blocking Deprotonation with Retention of Aromaticity in a Plant ent‐Copalyl Diphosphate Synthase Leads to Product Rearrangement

Substitution of a histidine, comprising part of the catalytic base group in the ent‐copalyl diphosphate synthases found in all seed plants for gibberellin phytohormone metabolism, by a larger aromatic residue leads to rearrangements. Through a series of 1,2‐hydride and methyl shifts of the initially formed bicycle predominant formation of (?)‐kolavenyl diphosphate is observed. Further mutational analysis and quantum chemical calculations provide mechanistic insight into the basis for this profound effect on product outcome.     

Preparation of “Si‐Centered” Chiral Silanes by Direct α‐Lithiation of Methylsilanes

The direct α‐lithiation of methyl‐substituted silanes as an efficient method for the preparation and elaboration of Si‐chiral compounds is reported. Deprotonation of chiral oligosilanes occurs selectively and with high yields at the methyl group of the stereogenic silicon center, even in the presence of multiple methylsilyl or methylgermyl substituents. Computational studies have confirmed this preference as a consequence of pre‐coordination of the lithiating agent by the amino side‐arm and repulsion effects in the corresponding transition state. This complexation is also obvious from X‐ray structure analyses of the α‐lithiated silanes, which exhibit intriguing structure formation patterns differing in the type of aggregation and the amount of alkyllithium used. An alternative route to Si‐chiral compounds is also presented, which involves desymmetrization of dimethylsilanes mediated by a chiral side‐arm. Structure analyses and computational studies have shown that the diastereoselectivity of this α‐lithiation is influenced by the selectivity of the formation of the stereogenic nitrogen upon complexation of the alkyllithium.     

The Structure of a Plant Tyrosinase from Walnut Leaves Reveals the Importance of “Substrate‐Guiding Residues” for Enzymatic Specificity

Tyrosinases and catechol oxidases are members of the class of type III copper enzymes. While tyrosinases accept both mono‐ and o‐diphenols as substrates, only the latter substrate is converted by catechol oxidases. Researchers have been working for decades to elucidate the monophenolase/diphenolase specificity on a structural level and have introduced an early hypothesis that states that the reason for the lack of monophenolase activity in catechol oxidases may be its structurally restricted active site. However, recent structural and biochemical studies of this enzyme class have raised doubts about this theory. Herein, the first crystal structure of a plant tyrosinase (from Juglans regia) is presented. The structure reveals that the distinction between mono‐ and diphenolase activity does not depend on the degree of restriction of the active site, and thus a more important role for amino acid residues located at the entrance to and in the second shell of the active site is proposed.     

Total Syntheses of (+)‐Sarcophytin, (+)‐Chatancin, (−)‐3‐Oxochatancin,and (−)‐Pavidolide B: A Divergent Approach

A concise and divergent approach for the total syntheses of four cembrane diterpenoids, namely (+)‐sarcophytin, (+)‐chatancin, (?)‐3‐oxochatancin, and (?)‐pavidolide B, has been developed, and it also led to the structural revision of (?)‐isosarcophytin. The key steps of the strategy feature a double Mukaiyama Michael addition/elimination, a Helquist annulation, two substrate‐controlled facial‐selective hydrations, and a pinacol rearrangement. The described syntheses not only achieved these natural products in an efficient manner, but also provided insight into the biosynthetic relationship between the two different skeletons.     

Structure and Biosynthesis of Xenoamicins from Entomopathogenic Xenorhabdus

During the search for novel natural products from entomopathogenic Xenorhabdus doucetiae DSM17909 and X. mauleonii DSM17908 novel peptides named xenoamicins were identified in addition to the already known antibiotics xenocoumacin and xenorhabdin. Xenoamicins are acylated tridecadepsipeptides consisting of mainly hydrophobic amino acids. The main derivative xenoamicin A ( 1 ) was isolated from X. mauleonii DSM17908, and its structure elucidated by detailed 1 D and 2 D NMR experiments. Detailed MS experiments, also in combination with labeling experiments, confirmed the determined structure and allowed structure elucidation of additional derivatives. Moreover, the xenoamicin biosynthesis gene cluster was identified and analyzed in X. doucetiae DSM17909, and its participation in xenoamicin biosynthesis was confirmed by mutagenesis. Advanced Marfey’s analysis of 1 showed that the absolute configuration of the amino acids is in agreement with the predicted stereochemistry deduced from the nonribosomal peptide synthetase XabABCD. Biological testing revealed activity of 1 against Plasmodium falciparum and other neglected tropical diseases but no antibacterial activity.