David James Miller
Cardiff University
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Natural Product Reports | 2012
David James Miller; Rudolf Konrad Allemann
Sesquiterpene synthases catalyse the metal dependent turnover of farnesyl diphosphate to generate a class of natural products characterised by an enormous diversity in structure, stereochemistry, biological function and application. It has been proposed that these enzymes take a passive role in the reactions they catalyse and that they serve mostly as stereochemical templates, within which the reactions take place. Here, recent research into the structure and function of sesquiterpene synthases and the mechanisms of the reactions that they catalyse will be reviewed to suggest that these fascinating enzymes play multifaceted active roles in what are arguably the most complex biosynthetic reactions.
Journal of Biological Chemistry | 2008
Ekaterina Y. Shishova; Fanglei Yu; David James Miller; Juan A. Faraldos; Yuxin X. Zhao; Robert M. Coates; Rudolf Konrad Allemann; David E. Cane; David W. Christianson
The universal sesquiterpene precursor, farnesyl diphosphate (FPP), is cyclized in an Mg2+-dependent reaction catalyzed by the tetrameric aristolochene synthase from Aspergillus terreus to form the bicyclic hydrocarbon aristolochene and a pyrophosphate anion (PPi) coproduct. The 2.1-Å resolution crystal structure determined from crystals soaked with FPP reveals the binding of intact FPP to monomers A-C, and the binding of PPi and Mg2+B to monomer D. The 1.89-Å resolution structure of the complex with 2-fluorofarnesyl diphosphate (2F-FPP) reveals 2F-FPP binding to all subunits of the tetramer, with Mg2+Baccompanying the binding of this analogue only in monomer D. All monomers adopt open activesite conformations in these complexes, but slight structural changes in monomers C and D of each complex reflect the very initial stages of a conformational transition to the closed state. Finally, the 2.4-Å resolution structure of the complex with 12,13-difluorofarnesyl diphosphate (DF-FPP) reveals the binding of intact DF-FPP to monomers A-C in the open conformation and the binding of PPi, Mg2+B, and Mg2+C to monomer D in a predominantly closed conformation. Taken together, these structures provide 12 independent “snapshots” of substrate or product complexes that suggest a possible sequence for metal ion binding and conformational changes required for catalysis.
ChemBioChem | 2007
David James Miller; Fanglei Yu; Rudolf Konrad Allemann
The mechanism of the conversion of (E,E)‐farnesyl diphosphate (FPP, 1 a) to aristolochene (6) catalyzed by aristolochene synthase from Penicillium roqueforti has been proposed to proceed through the neutral intermediate germacrene A (4 a). However, much of the experimental evidence is also in agreement with a mechanism in which germacrene A is not an intermediate in the predominant mechanism that leads to the formation of aristolochene, but rather an off‐pathway product that is formed in a side reaction. Hence, to elucidate the mechanism of FPP cyclisation the substrate analogue 2‐fluoroFPP (1 b) was synthesized, and upon incubation with aristolochene synthase was converted to a single pentane extractable product according to GC‐MS analysis. On the basis of NMR analyses this product was identified as 2‐fluorogermacrene A (4 b). Variable temperature 1H NMR spectroscopy indicated the existence of two conformers of 4 b that were in slow exchange at −60 °C, while at 90 °C the two isomers gave rise to averaged NMR signals. In the major isomer (∼75 %) the methyl groups on C3 and C7 were most likely in the down–down orientation as had been observed for other (E,E)‐germacranes. This work suggests that after an initial concerted cyclisation of FPP to germacryl cation deprotonation leads to the formation of germacrene A, and provides compelling evidence that germacrene A is indeed an on‐pathway product of catalysis by aristolochene synthase.
Biochemical Journal | 2013
Jian‑Xu Li; Xin Fang; Qin Zhao; Ju-Xin Ruan; Chang‑Qing Yang; Ling Jian Wang; David James Miller; Juan A. Faraldos; Rudolf Konrad Allemann; Xiao-Ya Chen; Peng Zhang
Most TPSs (terpene synthases) contain plasticity residues that are responsible for diversified terpene products and functional evolution, which provide a potential for improving catalytic efficiency. Artemisinin, a sesquiterpene lactone from Artemisia annua L., is widely used for malaria treatment and progress has been made in engineering the production of artemisinin or its precursors. In the present paper, we report a new sesquiterpene synthase from A. annua, AaBOS (A. annua α-bisabolol synthase), which has high sequence identity with AaADS (A. annua amorpha-4,11-diene synthase), a key enzyme in artemisinin biosynthesis. Comparative analysis of the two enzymes by domain-swapping and structure-based mutagenesis led to the identification of several plasticity residues, whose alteration changed the product profile of AaBOS to include γ-humulene as the major product. To elucidate the underlying mechanisms, we solved the crystal structures of AaBOS and a γ-humulene-producing AaBOS mutant (termed AaBOS-M2). Among the plasticity residues, position 399, located in the substrate-binding pocket, is crucial for both enzymes. In AaBOS, substitution of threonine for leucine (AaBOSL339T) is required for γ-humulene production; whereas in AaADS, replacing the threonine residue with serine (AaADST399S) resulted in a substantial increase in the activity of amorpha-4,11-diene production, probably as a result of accelerated product release. The present study demonstrates that substitution of plasticity residues has potential for improving catalytic efficiency of the enzyme.
Journal of the American Chemical Society | 2012
Juan A. Faraldos; David James Miller; Veronica Gonzalez; Zulfa Yoosuf-Aly; Oscar Cascón; Amang Li; Rudolf Konrad Allemann
Recombinant (+)-δ-cadinene synthase (DCS) from Gossypium arboreum catalyzes the metal-dependent cyclization of (E,E)-farnesyl diphosphate (FDP) to the cadinane sesquiterpene δ-cadinene, the parent hydrocarbon of cotton phytoalexins such as gossypol. In contrast to some other sesquiterpene cyclases, DCS carries out this transformation with >98% fidelity but, as a consequence, leaves no mechanistic traces of its mode of action. The formation of (+)-δ-cadinene has been shown to occur via the enzyme-bound intermediate (3R)-nerolidyl diphosphate (NDP), which in turn has been postulated to be converted to cis-germacradienyl cation after a 1,10-cyclization. A subsequent 1,3-hydride shift would then relocate the carbocation within the transient macrocycle to expedite a second cyclization that yields the cadinenyl cation with the correct cis stereochemistry found in (+)-δ-cadinene. An elegant 1,10-mechanistic pathway that avoids the formation of (3R)-NDP has also been suggested. In this alternative scenario, the final cadinenyl cation is proposed to be formed through the intermediacy of trans, trans-germacradienyl cation and germacrene D. In addition, an alternative 1,6-ring closure mechanism via the bisabolyl cation has previously been envisioned. We report here a detailed investigation of the catalytic mechanism of DCS using a variety of mechanistic probes including, among others, deuterated and fluorinated FDPs. Farnesyl diphosphate analogues with fluorine at C2 and C10 acted as inhibitors of DCS, but intriguingly, after prolonged overnight incubations, they yielded 2F-germacrene(s) and a 10F-humulene, respectively. The observed 1,10-, and to a lesser extent, 1,11-cyclization activity of DCS with these fluorinated substrates is consistent with the postulated macrocyclization mechanism(s) en route to (+)-δ-cadinene. On the other hand, mechanistic results from incubations of DCS with 6F-FPP, (2Z,6E)-FDP, neryl diphosphate, 6,7-dihydro-FDP, and NDP seem to be in better agreement with the potential involvement of the alternative biosynthetic 1,6-ring closure pathway. In particular, the strong inhibition of DCS by 6F-FDP, coupled to the exclusive bisabolyl- and terpinyl-derived product profiles observed for the DCS-catalyzed turnover of (2Z,6E)-farnesyl and neryl diphosphates, suggested the intermediacy of α-bisabolyl cation. DCS incubations with enantiomerically pure [1-(2)H(1)](1R)-FDP revealed that the putative bisabolyl-derived 1,6-pathway proceeds through (3R)-nerolidyl diphosphate (NDP), is consistent with previous deuterium-labeling studies, and accounts for the cis stereochemistry characteristic of cadinenyl-derived sesquiterpenes. While the results reported here do not unambiguously rule in favor of 1,6- or 1,10-cyclization, they demonstrate the mechanistic versatility inherent to DCS and highlight the possible existence of multiple mechanistic pathways.
Organic and Biomolecular Chemistry | 2008
David James Miller; Jiali Gao; Donald G. Truhlar; Neil James Young; Veronica Gonzalez; Rudolf Konrad Allemann
The aristolochene synthase catalysed cyclisation of farnesyl diphosphate (1) has been postulated to proceed through (S)-germacrene A (3). However, the active site acid that reprotonates this neutral intermediate has so far proved difficult to identify and, based on high level ab initio molecular orbital and density functional theory calculations, a proton transfer mechanism has recently been proposed, in which proton transfer from C12 of germacryl cation to the C6,C7-double bond of germacryl cation (2) proceeds either directly or via a tightly bound water molecule. In this work, the stereochemistry of the elimination and protonation reactions was investigated by the analysis of the reaction products from incubation of 1 and of [12,12,12,13,13,13-(2)H(6)]-farnesyl diphosphate (15) with aristolochene synthase from Penicillium roqueforti (PR-AS) in H(2)O and D(2)O. The results reveal proton loss from C12 during the reaction and incorporation of another proton from the solvent. Incubation of with PR-AS in D(2)O led to the production of (6R)-[6-(2)H] aristolochene, indicating that protonation occurs from the face of the 10-membered germacrene ring opposite the isopropylidene group. Hence these results firmly exclude proton transfer from C12 to C6 of germacryl cation. We propose here Lys 206 as the general acid/base during PR-AS catalysis. This residue is part of a conserved network of hydrogen bonds, along which protons could be delivered from the solvent to the active site.
Tetrahedron Letters | 1998
Duane Stones; David James Miller; Martin W. Beaton; Trevor J. Rutherford; David Gani
A simple and efficient method for monitoring and quantifying the extent of loading onto polymer resin supports for solid-phase synthesis using 19F gel-phase NMR spectroscopy is described. This assay was utilised in the synthesis of an inositol monophosphatase inhibitor on Merrifield resin. A series of Merrifield resin derived benzylic ethers were prepared and were cleaved from the resin when treated with SnCl4 at room temperature to give the expected alcohol, phenol or olefin. This new cleavage method was used to remove the inositol monophosphatase inhibitor from the resin. A method for quantifying polymer support loading, using 19F gel phase NMR spectroscopy and a facile method for the cleavage of Merrifield resin derivatives is described in the context of a solid phase synthesis of phosphatase inhibitor 6.
Chemical Communications | 2012
Oscar Cascón; Sabrina Touchet; David James Miller; Veronica Gonzalez; Juan A. Faraldos; Rudolf Konrad Allemann
A small library of novel germacrenes was generated using a combination of two plant enzymes, germacrene A synthase, and D synthase and modified farnesyl diphosphate (FDP) analogues. This chemoenzymatic approach allows the preparation of potentially valuable volatiles for biological studies.
MedChemComm | 2012
Sarah Elizabeth Adams; Christian Parr; David James Miller; Rudolf Konrad Allemann; Maurice Bartlett Hallett
Calpain-1 is a Ca2+-activated cytosolic cysteine protease. Its activation has been linked to extravasation into inflamed tissue of white blood cells, especially neutrophils. Calpain-1 may therefore be an anti-inflammatory target for therapeutic intervention. 24 novel monohalogenated phenyl and indole mercaptoacrylic acid derivatives were synthesised. The location and nature of the ring-coupled halides strongly influenced the potency of these compounds to inhibit calpain activation. Several of the calpain-1 inhibitors showed IC50 values in the low nanomolar range and prevented cell shape change of neutrophils, a necessary prelude to their migration from the blood in the body.
Mini-reviews in Medicinal Chemistry | 2007
David James Miller; Rudolf Konrad Allemann
myo-Inositol monophosphatase has long been a target for drug discovery. Recent work has given detailed insight into its mechanism and dynamics plus new activities and some novel series of inhibitors. The goal of a bio-available inhibitor for this enzyme, however, remains a major challenge for medicinal chemistry.