RESUMEN
A computational modeling/protein engineering approach was applied to probe H234, C457, T509, Y510, and W587 within Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7), which spatially affects the C-10 cation of lanosterol formation. Substitution of Trp587 to aromatic residues supported the "aromatic hypothesis" that the π-electron-rich pocket is important for the stabilization of electron-deficient cationic intermediates. The Cys457 to Gly and Thr509 to Gly mutations disrupted the pre-existing H-bond to the protonating Asp456 and the intrinsic His234 : Tyr510 H-bond network, respectively, and generated achilleol A as the major product. An H234W/Y510W double mutation altered the ERG7 function to achilleol A synthase activity and generated achilleol A as the sole product. These results support the concept that a few-ring triterpene synthase can be derived from polycyclic cyclases by reverse evolution, and exemplify the power of computational modeling coupled with protein engineering both to study the enzyme's structure-function-mechanism relationships and to evolve new enzymatic activity.
Asunto(s)
Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Triterpenos/metabolismo , Sustitución de Aminoácidos , Transferasas Intramoleculares/química , Modelos Moleculares , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
A Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase mutant, ERG7(T384Y/Q450H/V454I), produced parkeol but not lanosterol as the sole end product. Parkeol undergoes downstream metabolism to generate compounds 9 and 10. In vitro incubation of parkeol produced a product profile similar to that of the in vivo experiment. In summary, parkeol undergoes a metabolic pathway similar to that of cycloartenol in yeast but distinct from that of lanosterol in yeast, suggesting that two different metabolic pathways of postoxidosqualene cyclization may exist in S. cerevisiae.
Asunto(s)
Transferasas Intramoleculares/biosíntesis , Saccharomyces cerevisiae/metabolismo , Ciclización , Transferasas Intramoleculares/química , Transferasas Intramoleculares/genética , Modelos Moleculares , Estructura Molecular , Mutación , Ingeniería de Proteínas , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Escualeno/análogos & derivados , Escualeno/química , Escualeno/metabolismoRESUMEN
Site-saturated substitution in Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase at Ile705 position produced three chair-boat-chair (C-B-C) truncated tricyclic compounds, two 17α-exocyclic protosteryl intermediates, two protosteryl C-17 truncated rearranged intermediates and the normal biosynthetic product, lanosterol. These results indicated the importance of the Ile705 residue in affecting lanosterol's C/D ring stabilization including 6-6-5 tricyclic and protosteryl C-17 cations and 17α/ß-exocyclic side chain stereochemistry.
Asunto(s)
Transferasas Intramoleculares/metabolismo , Lanosterol/química , Mutación , Saccharomyces cerevisiae/enzimología , Ciclización , Transferasas Intramoleculares/genética , Isoleucina/genética , Isoleucina/metabolismo , Lanosterol/metabolismo , Modelos Moleculares , Estereoisomerismo , Especificidad por SustratoRESUMEN
The Saccharomyces cerevisiae ERG7(Phe699) mutants produced one chair-chair-chair (C-C-C) and two chair-boat-chair (C-B-C) truncated tricyclic compounds, one tetracyclic 17alpha-exocyclic unrearranged intermediate, and two 17beta-exocyclic truncated rearranged intermediates. These results provided direct evidence for the importance of the residue in affecting mechanistic transitions between C-B-C and C-C-C substrate conformation and between the 17alpha- and 17beta-exocyclic side chain stereochemistry as well as in stabilizing the 6-6-5 tricyclic and the protosteryl C-17 cations.
Asunto(s)
Transferasas Intramoleculares/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Catálisis , Ciclización , Transferasas Intramoleculares/química , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Fenilalanina/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
A contact mapping strategy was applied to identify putative amino acid residues that influence the oxidosqualene-lanosterol B-ring cyclization reaction. A bicyclic intermediate with two altered deprotonation products, in conjunction with lanosterol, were isolated from the ERG7(Y707X) mutants, indicating that the Tyr707 residue may play a functional role in stabilizing the chair-boat bicyclic C-8 cation and the lanosteryl C-8/C-9 cation intermediates.