Functional Characterization of Oxidosqualene Cyclases Using Mutagenesis Approaches, Inhibition Studies, and SELEX Technology

• Main Authors: Cheng-Hsiang Chang, 張程翔
• Other Authors: Tung-Kung Wu
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/99647882297695039436

博士 === 國立交通大學 === 生物科技系所 === 96 === Abstract Oxidosqualene cyclases (EC 5.4.99-) constitute a family of enzymes that catalyze diverse cyclization/rearrangement reactions of (3S)-2,3-oxidosqualene (OS) into a distinct array of sterols and triterpenes. Notably, the product specificity among most of cyclase enzymes is species-dependent. The relationship between cyclization mechanism and enzymatic structure is extremely complex and attractive. In order to further elucidate the cyclization/rearrangement reaction of oxidosqualene cyclases, experiments including different molecular biological mutagenesis approaches as well as the bioorganic studies were accordingly carried out. First, the alanine-scanning mutagenesis and site-directed mutagenesis coupled with in vivo plasmid shuffling selection were employed to identify the catalytic or structural important residues in oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae (S. cerevisiae ERG7). Among the investigated sequence segment from Thr-509 to Ile-513, Tyr-510 showed the catalytic discrepancy in the cyclase activity upon mutagenic effect. The yeast transformant failed to complement the cyclase-deficiency when this position was mutated to tryptophan or lysine residues, but still maintained the yeast viability in the S. cerevisiae ERG7Y510A mutant. After analysis of the nonsaponifiable lipid from S. cerevisiae ERG7Y510A, the monocyclic achilleol A, tetracyclic lanosterol and parkeol were identified. Moreover, two monocyclic compounds, achilleol A and camelliol C, were isolated from the lethal S. cerevisiae ERG7Y510K and S. cerevisiae ERG7Y510W mutants. In order to further investigate the mutated effects on this residue, the site-saturated mutagenesis was subsequently performed. Diverse products including monocyclic, tricyclic, and different tetracyclic products were isolated from the S. cerevisiae ERG7Y510X mutants. Moreover, the inherent influence on product specificity via an altered coordinative interaction between the hypothesized catalytic dyad, Tyr-510 and His-234, were further examined in more detail via construction and analysis of a different set of S. cerevisiae ERG7H234X/Y510X double mutations. Moreover, other catalytically important residues and their respective premature cyclization products, involved in different cyclization/rearrangement stages, were also discovered by using similarly executed site-saturated mutagenesis, coupled with bioorganic characterization. In order to carefully explore the importance of these crucial residues within the enzymatic active site, plausible homology modeling structures were subsequently created. The diverse array of product profiles which were isolated from various mutated S. cerevisiae ERG7 cyclases was broadly representative. Moreover, the products’ tendency in different mutated enzymes was consequently understood by using the quantum mechanics calculation of Gaussian 03. In addition, the combination of chimeric enzyme library between Saccharomyces cerevisiae lanosterol synthase and Arabidopsis thaliana cycloartenol synthase was also constructed to determine the critical functional domain responsible for the product specificity. Ten diverse domain swapping chimeras were successfully created, and their activities were subsequently confirmed via plasmid shuffling selection. No divergence of the nonsaponifiable lipid patterns was observed among these inactive chimeras, suggesting that the rough partition might disrupt the enzyme structure. After comparison with the previous experimental results from the triterpene synthases, the product specificity-determining residues among these sterol-biosynthetic cyclases might be determined by just several functional crucial residues within the enzymatic active site. In parallel to the ongoing molecular biology approaches, we also performed a number of biochemical studies, including chromatographic purification, bioorganic characterization, and inhibition studies to examine the structure-function relationships for mammalian lanosterol cyclase. After successful purification and tandem mass characterization of bovine liver OSC, the gene encoding bovine liver OSC was subsequently determined. The deduced amino acid sequence showed >80% identity to that of the other three mammalian lanosterol synthases. The bovine liver OSC gene was also successfully cloned and functionally expressed in a yeast erg7 disruption strain. Moreover, in order to better understand the inhibiting mechanism of one potent OSC inhibitor, Ro48-8071, as well as to solve the exact inhibitor binding site, the photoaffinity labeling and chemical fluorescent modification of Ro48-8071 was also carried out. Several Ro48-8071-based fluorescent probes were developed and their inhibitory activity or fluorescence characteristics were analyzed. The results of chemical modification of Ro48-8071 suggested that the fluorescent Ro48-8071 derivatives dramatically reduced its inhibitory activity for purified bovine liver OSC. Moreover, the interactions between fluorescent Ro48-8071 derivative and the active site of bovine liver OSC, as well as the orientation of these probes have obviously changed, based on the molecular docking experimental data. In the future, improved site-specific fluorescent probes should be developed and applied to the chemical proteomic field for effectively screening the OSC drugs. By another approach, a randomly selected combinatorial approach, SELEX, was utilized for screening the potential OSC-binding aptamer molecules of the bovine liver OSC. After nine rounds of SELEX screening, a diverse array of aptamer candidates was isolated from the single-strand DNA library. These aptamer molecules exhibited the definitive interaction with the targeted protein and also revealed the approximate nM range affinity for bovine liver OSC. However, the binding interaction between individual aptamers and the cyclase protein should be explored in the future. These obtained OSC-binding aptamers will be applied in the pharmaceutical or diagnostic applications for lanosterol synthase as well as for studies in the cholesterol pathway in the future. Thus, the combination of results obtained from the molecular biological approaches, including alanine-scanning, site-directed/saturated mutagenesis, domain swapping experiments, homology modeling structure, and quantum mechanics calculation, a better understanding of the cyclization/rearrangement mechanism of oxidosqualene cyclase has been achieved. In addition, the biochemical characterization coupled with the bioorganic studies toward mammalian lanosterol synthase provided valuable information, especially in obtaining the purified cyclase protein, and illustrating the inhibition mechanism of Ro48-8071. Moreover, the in vitro SELEX procedure opens new avenues in rational designing of antifungal agents and hypocholesteremic agents.