| Summary: | 碩士 === 國立交通大學 === 生物科技系所 === 93 === Oxidosqualene cyclases (OSCs) constitute a family of enzymes which catalyze the conversion of linear oxidosqualene into polycyclic tetracyclic and pentacyclic triterpenoids in a one-step enzyme-catalyzed reaction. The OSC-catalyzed cyclization /rearrangement reaction represents one of the most remarkable and fascinating biotransformations found in nature. In order to clarify the function of the amino acid residues located on the putative active site cavity surface of oxidosqualene-lanosterol cyclase of yeast Saccharomyces cerevisiae, site-directed mutagenesis coupled with plasmid shuffle experiments were carried out. Following the assaying of twenty-nine alanine-scanning mutants for the complementation of yeast viability, nine inactive mutants(OSCF104T, OSCH146A, OSCW232A, OSCW232R, OSCH234A, OSCM532A, OSCW587A, OSCF699A and OSCY707A) were further analyzed for product profile. Further product isolation and characterization of each mutant showed that OSCW232A mutant produced Protosta-12,24-dien-3ß-ol, Lanosterol and Parkeol as end products, exhibiting the same product profile as that of OSCH234Y mutant. In parallel, the OSCH234A mutant produced Protosta-12,24-dien-3ß-ol, Lanosterol, Parkeol, and Protosta-20,24-dien -3ß-ol as end products.
To completely characterize the functional role of W232 in OSC activity, the saturated mutagenesis experiments at W232 position were performed. Following the analysis of each mutant products, two additional new products with retention time (rt.) at 26.0 and 26.5 min were observed on GC-MS spectrometry. The EI low mass spectrum of rt. 26 min product showed a molecular ion at m/z 426 and fragment peaks at 357 ([M-C5H9]+) and 313 ([M-C8H15-H2]+), suggesting an intact tetracyclic ring. In summary, the results suggest a role for W232 in stabilizing a cationic intermediate, probably after protosteryl cation formation, and in determining the deprotonation position for diverse product profile.
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