Astertarone A Synthase from Chinese Cabbage Does Not Produce the C4-Epimer: Mechanistic Insights.

The Brassica rapa oxidosqualene cyclase Bra032185 makes 60% astertarone A (1) and 20 minor triterpenes (0.1-11%). Mechanistic analysis indicates the absence of an enol intermediate to generate the 4 S epimer of 1, unless workup involves saponification. Bra032185 and its closest Arabidopsis thaliana homologue diverged markedly in product profiles over a short evolutionary distance, while developing opposite C18 configurations. Active-site residue comparison of Bra032185 with friedelin and shionone synthases suggests convergent evolution to 3-ketotriterpenes.

Are Isoursenol and γ-Amyrin Rare Triterpenes in Nature or Simply Overlooked by Usual Analytical Methods?

Among pentacyclic triterpenes commonly found in plants, γ-amyrin and isoursenol are seldom reported and considered rare in nature. It was hypothesized that these triterpenes are instead routinely overlooked due to inadequate spectral characterization. γ-Amyrin was prepared by HCOOH isomerization of α-amyrin, and isoursenol was isolated from products of a heterologously expressed oxidosqualene cyclase. With precise NMR and GC-MS data, a metabolomics strategy was used to identify isoursenol and γ-amyrin in a wide range of plants.

Mixed bioengineering-chemical synthesis approach for the efficient preparation of Δ7-dafachronic acid.

Combining bioengineering with chemical synthesis has enabled an efficient method for producing Δ7-dafachronic acid, a steroidal hormone associated with nematode germline longevity. Saccharomyces cerevisiae was engineered to produce 7,24-cholestadienol, a convenient starting material for a four-step synthesis of Δ7-dafachronic acid.

An effective strategy for exploring unknown metabolic pathways by genome mining.

Plants allocate an estimated 15-25% of their proteome to specialized metabolic pathways that remain largely uncharacterized. Here, we describe a genome mining strategy for exploring such unknown pathways and demonstrate this approach for triterpenoids by functionally characterizing three cytochrome P450s from Arabidopsis thaliana . Building on proven methods for characterizing oxidosqualene cyclases, we heterologously expressed in yeast known cyclases with candidate P450s chosen from gene clustering and microarray coexpression patterns. The yeast cultures produced mg/L amounts of plant metabolites in vivo without the complex phytochemical background of plant extracts. Despite this simplification, the product multiplicity and novelty overwhelmed analytical efforts by MS methods. HSQC analysis overcame this problem. Side-by-side HSQC comparisons of crude P450 extracts against a control resolved even minor P450 products among ~100 other yeast metabolites spanning a dynamic range of >10,000:1. HSQC and GC-MS then jointly guided purification and structure determination by classical NMR methods. Including our present results for P450 oxidation of thalianol, arabidiol, and marneral, the metabolic fate for most of the major triterpene synthase products in Arabidopsis is now at least partially known.

Product profile of PEN3: the last unexamined oxidosqualene cyclase in Arabidopsis thaliana.

The triterpene product profile is reported for At5g36150 (PEN3), the last unexamined oxidosqualene cyclase in the reference plant Arabidopsis thaliana. PEN3 makes tirucalla-7,24-dien-3beta-ol ( approximately 85%) and several minor products. Also discussed are the unexpectedly facile convergent evolution of another Arabidopsis tirucalladienol synthase (LUP5), mechanistic origins of the 20S configuration, and active-site remodeling necessary to accommodate the 17alpha side chain. This work marks the first completed functional characterization of all triterpene synthases in a higher plant.

Protostadienol biosynthesis and metabolism in the pathogenic fungus Aspergillus fumigatus.

Details of the fungal biosynthetic pathway to helvolic acid and other fusidane antibiotics remain obscure. During product characterization of oxidosqualene cyclases in Aspergillus fumigatus, we found the long-sought cyclase that makes (17Z)-protosta-17(20),24-dien-3beta-ol, the precursor of helvolic acid. We then identified a gene cluster encoding the pathway to helvolic acid, which is controlled by a transcription regulator (LaeA) associated with fungal virulence. Evidence regarding the evolutionary origin and taxonomic distribution of fusidane biosynthesis is also presented.

Trinorlupeol: a major nonsterol triterpenoid in Arabidopsis.

We report the structure determination of 20,29,30-trinorlup-18-en-3beta-ol (trinorlupeol) and establish this novel C 27 metabolite as a major nonsterol triterpenoid in Arabidopsis thaliana. Trinorlupeol was concentrated in cuticular waxes, notably in the plant stem, floral buds, and seedpods, but not in leaves. Based on expression data and functional characterization of A. thaliana oxidosqualene cyclases, we propose that LUP1 is the cyclase responsible for trinorlupeol biosynthesis. Also described are two oxidized trinorlupeols and additional biosynthetic insights.

Arabidopsis camelliol C synthase evolved from enzymes that make pentacycles.

We establish by heterologous expression that the Arabidopsis thaliana oxidosqualene cyclase At1g78955 (CAMS1) makes camelliol C (98%), achilleol A (2%), and beta-amyrin (0.2%). CAMS1 is the first characterized cyclase that generates predominantly a monocyclic triterpene alcohol. Phylogenetic analysis shows that CAMS1 evolved from enzymes that make pentacycles, thus revealing that its pentacyclic beta-amyrin byproduct is an evolutionary relic. Sequence alignments support prior suggestions that decreased steric bulk at a key active-site residue promotes monocycle formation.

An oxidosqualene cyclase makes numerous products by diverse mechanisms: a challenge to prevailing concepts of triterpene biosynthesis.

The genome of the model plant Arabidopsis thaliana encodes 13 oxidosqualene cyclases, 9 of which have been characterized by heterologous expression in yeast. Here we describe another cyclase, baruol synthase (BARS1), which makes baruol (90%) and 22 minor products (0.02-3% each). This represents as many triterpenes as have been reported for all other Arabidopsis cyclases combined. By accessing an extraordinary repertoire of mechanistic pathways, BARS1 makes numerous skeletal types and deprotonates the carbocation intermediates at 14 different sites around rings A, B, C, D, and E. This undercurrent of structural and mechanistic diversity in a superficially accurate enzyme is incompatible with prevailing concepts of triterpene biosynthesis, which posit tight control over the mechanistic pathway through cation-pi interactions, with a single proton acceptor in a hydrophobic active site. Our findings suggest that mechanistic diversity is the default for triterpene biosynthesis and that product accuracy results from exclusion of alternative pathways.

Stereochemistry of water addition in triterpene synthesis: the structure of arabidiol.

An oxidosqualene cyclase from Arabidopsis thaliana makes arabidiol, a tricyclic triterpene reported with indeterminate side-chain stereochemistry. We established the full structure of arabidiol through chemical degradation, NOE experiments, and molecular modeling. By examining the mechanistic constraints that govern water addition in triterpene synthesis, we further show how the stereochemistry of hydroxylation can generally be deduced a priori, why deprotonation is more common than hydroxylation, and why cyclases that perform hydroxylation also generate olefinic byproducts.

Arabidopsis thaliana squalene epoxidase 1 is essential for root and seed development.

Squalene epoxidase converts squalene into oxidosqualene, the precursor of all known angiosperm cyclic triterpenoids, which include membrane sterols, brassinosteroid phytohormones, and non-steroidal triterpenoids. In this work, we have identified six putative Arabidopsis squalene epoxidase (SQE) enzymes and used heterologous expression in yeast to demonstrate that three of these enzymes, SQE1, SQE2, and SQE3, can epoxidize squalene. We isolated and characterized Arabidopsis sqe1 mutants and discovered severe developmental defects, including reduced root and hypocotyl elongation. Adult sqe1-3 and sqe1-4 plants have diminished stature and produce inviable seeds. The sqe1-3 mutant accumulates squalene, consistent with a block in the triterpenoid biosynthetic pathway. Therefore, SQE1 function is necessary for normal plant development, and the five SQE-like genes remaining in this mutant are not fully redundant with SQE1.

Mechanistic insights into triterpene synthesis from quantum mechanical calculations. Detection of systematic errors in B3LYP cyclization energies.

Most quantum mechanical studies of triterpene synthesis have been done on small models. We calculated mPW1PW91/6-311+G(2d,p)//B3LYP/6-31G* energies for many C30H51O+ intermediates to establish the first comprehensive energy profiles for the cationic cyclization of oxidosqualene to lanosterol, lupeol, and hopen-3beta-ol. Differences among these 3 profiles were attributed to ring strain, steric effects, and proton affinity. Modest activation energy barriers and the ample exothermicity of most annulations indicated that the cationic intermediates rarely need enzymatic stabilization. The course of reaction is guided by hyperconjugation of the carbocationic 2p orbital with parallel C-C and C-H bonds. Hyperconjugation for cations with a horizontal 2p orbital (in the plane of the ABCD ring system) leads to annulation and ring expansion. If the 2p orbital becomes vertical, hyperconjugation fosters 1,2-methyl and hydride shifts. Transition states leading to rings D and E were bridged cyclopropane/carbonium ions, which allow ring expansion/annulation to bypass formation of undesirable anti-Markovnikov cations. Similar bridged species are also involved in many cation rearrangements. Our calculations revealed systematic errors in DFT cyclization energies. A spectacular example was the B3LYP/6-311+G(2d,p)//B3LYP/6-31G* prediction of endothermicity for the strongly exothermic cyclization of squalene to hopene. DFT cyclization energies for the 6-311+G(2d,p) basis set ranged from reasonable accuracy (mPW1PW91, TPSSh with 25% HF exchange) to underestimation (B3LYP, HCTH, TPSS, O3LYP) or overestimation (MP2, MPW1K, PBE1PBE). Despite minor inaccuracies, B3LYP/6-31G* geometries usually gave credible mPW1PW91 single-point energies. Nevertheless, DFT energies should be used cautiously until broadly reliable methods are established.

Lanosterol biosynthesis in plants.

Plants biosynthesize sterols from cycloartenol using a pathway distinct from the animal and fungal route through lanosterol. Described herein are genome-mining experiments revealing that Arabidopsis encodes, in addition to cycloartenol synthase, an accurate lanosterol synthase (LSS)--the first example of lanosterol synthases cloned from a plant. The coexistence of cycloartenol synthase and lanosterol synthase implies specific roles for both cyclopropyl and conventional sterols in plants. Phylogenetic reconstructions reveal that lanosterol synthases are broadly distributed in eudicots but evolved independently from those in animals and fungi. Novel catalytic motifs establish that plant lanosterol synthases comprise a third catalytically distinct class of lanosterol synthase.

A putative precursor of isomalabaricane triterpenoids from lanosterol synthase mutants.

[reaction: see text]. Known lanosterol synthase mutants produce monocyclic or tetracyclic byproducts from oxidosqualene. We describe Erg7 Tyr510 mutants that cause partial substrate misfolding and generate a tricyclic byproduct. This novel triterpene, (13alphaH)-isomalabarica-14(27),17E,21-trien-3beta-ol, is the likely biosynthetic precursor of isomalabaricane triterpenoids in sponges. The results suggest the facile evolution of protective triterpenoids in sessile animals.

Enzymatic cyclization of dioxidosqualene to heterocyclic triterpenes.

Oxidosqualene cyclases normally produce triterpenes from 2,3-(S)-oxidosqualene (OS) but also can cyclize its minor companion (3S,22S)-2,3:22,23-dioxidosqualene (DOS). We explored DOS cyclization in plant triterpene synthesis using a recombinant lupeol synthase (LUP1) heterologously expressed in yeast. Incubation of LUP1 with 3S,22S-DOS gave epoxydammaranes epimeric at C20 and a 17,24-epoxybaccharane in a 4:2:3 ratio. The products reflected a new mechanistic paradigm for DOS cyclization. The structures were determined by NMR and GC-MS, and recent errors in the epoxydammarane literature were rectified. Some DOS metabolites are likely candidates for regulating triterpenoid biosynthesis, while others may be precursors of saponin aglycones. Our in vivo experiments in yeast generated substantial amounts of DOS metabolites in a single enzymatic step, suggesting a seminal role for the DOS shunt pathway in the evolution of saponin synthesis. Quantum mechanical calculations revealed oxonium ion intermediates, whose reactivity altered the usual mechanistic patterns of triterpene synthesis. Further analysis indicated that the side chain of the epoxydammarenyl cation intermediate is in an extended conformation. The overall results establish new roles for DOS in triterpene synthesis and exemplify how organisms can increase the diversity of secondary metabolites without constructing new enzymes.

Enzyme redesign: two mutations cooperate to convert cycloartenol synthase into an accurate lanosterol synthase.

Efforts to modify the catalytic specificity of enzymes consistently show that it is easier to broaden the substrate or product specificity of an accurate enzyme than to restrict the selectivity of one that is promiscuous. Described herein are experiments in which cycloartenol synthase was redesigned to become a highly accurate lanosterol synthase. Several single mutants have been described that modify the catalytic specificity of cycloartenol to form some lanosterol. Modeling studies were undertaken to identify combinations of mutations that cooperate to decrease the formation of products other than lanosterol. A double mutant was constructed and characterized and was shown to cyclize oxidosqualene accurately to lanosterol (99%). This catalytic change entailed both relocating polarity with a His477Asn mutation and modifying steric constraints with an Ile481Val mutation.

Structure and reactivity of the dammarenyl cation: configurational transmission in triterpene synthesis.

[reaction: see text] The dammarenyl cation (13) is the last common intermediate in the cyclization of oxidosqualene to a diverse array of secondary triterpene metabolites in plants. We studied the structure and reactivity of 13 to understand the factors governing the regio- and stereospecificity of triterpene synthesis. First, we demonstrated that 13 has a 17beta side chain in Arabidopsis thaliana lupeol synthase (LUP1) by incubating the substrate analogue (18E)-22,23-dihydro-20-oxaoxidosqualene (21) with LUP1 from a recombinant yeast strain devoid of other cyclases and showing that the sole product of 21 was 3beta-hydroxy-22,23,24,25,26,27-hexanor-17beta-dammaran-20-one. Quantum mechanical calculations were carried out on gas-phase models to show that the 20-oxa substitution has negligible effect on substrate binding and on the activation energies of reactions leading to either C17 epimer of 13. Further molecular modeling indicated that, because of limited rotational freedom in the cyclase active site cavity, the C17 configuration of the tetracyclic intermediate 13 can be deduced from the angular methyl configuration of the pentacyclic or 6-6-6-6 tetracyclic product. This rule of configurational transmission aided in elucidating the mechanistic pathway accessed by individual cyclases. Grouping of cyclases according to mechanistic and taxonomic criteria suggested that the transition between pathways involving 17alpha and 17beta intermediates occurred rarely in evolutionary history. Two other mechanistic changes were also rare, whereas variations on cation rearrangements evolved readily. This perspective furnished insights into the phylogenetic relationships of triterpene synthases.

Cholesterol import by Aspergillus fumigatus and its influence on antifungal potency of sterol biosynthesis inhibitors.

High mortality rates from invasive aspergillosis in immunocompromised patients are prompting research toward improved antifungal therapy and better understanding of fungal physiology. Herein we show that Aspergillus fumigatus, the major pathogen in aspergillosis, imports exogenous cholesterol under aerobic conditions and thus compromises the antifungal potency of sterol biosynthesis inhibitors. Adding serum to RPMI medium led to enhanced growth of A. fumigatus and extensive import of cholesterol, most of which was stored as ester. Growth enhancement and sterol import also occurred when the medium was supplemented with purified cholesterol instead of serum. Cells cultured in RPMI medium with the sterol biosynthesis inhibitors itraconazole or voriconazole showed retarded growth, a dose-dependent decrease in ergosterol levels, and accumulation of aberrant sterol intermediates. Adding serum or cholesterol to the medium partially rescued the cells from the drug-induced growth inhibition. We conclude that cholesterol import attenuates the potency of sterol biosynthesis inhibitors, perhaps in part by providing a substitute for membrane ergosterol. Our findings establish significant differences in sterol homeostasis between filamentous fungi and yeast. These differences indicate the potential value of screening aspergillosis antifungal agents in serum or other cholesterol-containing medium. Our results also suggest an explanation for the antagonism between itraconazole and amphotericin B, the potential use of Aspergillus as a model for sterol trafficking, and new insights for antifungal drug development.