RESUMO
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.
Assuntos
Antibacterianos/isolamento & purificação , Aspergillus fumigatus , Ácido Fusídico/análogos & derivados , Transferases Intramoleculares/metabolismo , Triterpenos/isolamento & purificação , Antibacterianos/química , Antibacterianos/farmacologia , Aspergillus fumigatus/química , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Ácido Fusídico/química , Ácido Fusídico/isolamento & purificação , Ácido Fusídico/farmacologia , Transferases Intramoleculares/genética , Estrutura Molecular , Estereoisomerismo , Triterpenos/químicaRESUMO
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.
Assuntos
Transferases Intramoleculares/metabolismo , Triterpenos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Transferases Intramoleculares/química , Estrutura Molecular , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismo , Triterpenos/químicaRESUMO
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.
Assuntos
Arabidopsis/química , Arabidopsis/metabolismo , Transferases Intramoleculares/química , Transferases Intramoleculares/metabolismo , Lanosterol/biossíntese , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Mapeamento Cromossômico , Transferases Intramoleculares/genética , Proteínas de Plantas/genéticaRESUMO
[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.
Assuntos
Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Poríferos/química , Triterpenos , Animais , Evolução Biológica , Contraindicações , Estrutura Molecular , Mutação , Esqualeno/análogos & derivados , Esqualeno/química , Esqualeno/metabolismo , Triterpenos/química , Triterpenos/isolamento & purificaçãoRESUMO
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.
Assuntos
Esqualeno/análogos & derivados , Triterpenos/síntese química , Ciclização , Transferases Intramoleculares/química , Transferases Intramoleculares/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Esqualeno/química , Esqualeno/metabolismo , Triterpenos/metabolismoRESUMO
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.
Assuntos
Transferases Intramoleculares/química , Engenharia de Proteínas/métodos , Animais , Arabidopsis/enzimologia , Catálise , Ativação Enzimática/genética , Transferases Intramoleculares/genética , Modelos Moleculares , Conformação Molecular , Mutagênese , Conformação Proteica , Esqualeno/análogos & derivados , Esqualeno/síntese química , Esqualeno/químicaRESUMO
[reaction: see text] Cycloartenol synthase cyclizes and rearranges oxidosqualene to the protosteryl cation and then specifically deprotonates from C-19. To identify mutants that deprotonate differently, randomly generated mutant cycloartenol synthases were selected in a yeast lanosterol synthase mutant. A novel His477Asn mutant was uncovered that produces 88% lanosterol and 12% parkeol. The His477Gln mutant produces 73% parkeol, 22% lanosterol, and 5% Delta(7)-lanosterol. These are the most accurate lanosterol synthase and parkeol synthase that have been generated by mutagenesis.