Browsing by Author "Liu, Yuan-Ting"

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Alteration of the Substrate's Prefolded Conformation and Cyclization Stereochemistry of Oxidosqualene-Lanosterol Cyclase of Saccharomyces cerevisiae by Substitution at Phenylalanine 699
(2010-02-05)
Wu, Tung-Kung
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Chang, Cheng-Hsiang
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Wen, Hao-Yu
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Liu, Yuan-Ting
;
Li, Wen-Hsuan
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 17 alpha-exocyclic unrearranged intermediate, and two 17 beta-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 17 alpha- and 17 beta-exocyclic side chain stereochemistry as well as in stabilizing the 6 -6-5 tricyclic and the protosteryl C-17 cations.
Differential Stereocontrolled Formation of Tricyclic Triterpenes by Mutation of Tyrosine 99 of the Oxidosqualene-Lanosterol Cyclase from Saccharomyces cerevisiae
(2009-11-01)
Wu, Tung-Kung
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Li, Wen-Hsuan
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Chang, Cheng-Hsiang
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Wen, Hao-Yu
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Liu, Yuan-Ting
The function of the Tyr99 residue from Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7) was analyzed by constructing deletion and site-saturated mutants. Two truncated intermediates, (13 alpha H)-isomalabarica-14Z,17E,21-trien-3 beta-ol and (13 alpha H)-isomalabarica-14E,17E,21-trien-3 beta-ol, were isolated from the ERG7(Y99X) mutants. These results suggest that the functional role of ERG7(Y99) is to affect both chair-boat 6-6-5 tricyclic Markovnikov C-14 cation stabilization and the stereochemistry of the protons at the C-15 position for subsequent deprotonation. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)
Diversity of sugar acceptor of glycosyltransferase 1 from Bacillus cereus and its application for glucoside synthesis
(2016-05)
Chiu, Hsi-Ho
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Shen, Mo-Yuan
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Liu, Yuan-Ting
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Fu, Yu-Lieh
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Chiu, Yu-An
Glycosyltransferase 1 from Bacillus cereus (BcGT1) catalyzes the transfer of a glucosyl moiety from uridine diphosphate glucose (UDP-glucose) to various acceptors; it was expressed and characterized. The specificity of acceptors was found to be broad: more than 20 compounds classified into O-, S-, and N-linkage glucosides can be prepared with BcGT1 catalysis. Based on this work, we conclude that the corresponding acceptors of these compounds must possess the following features: (1) the acceptors must contain at least one aromatic or fused-aromatic or heteroaromatic ring; (2) the reactive hydroxyl or sulfhydryl or amino group can attach either on the aromatic ring or on its aliphatic side chain; and (3) the acceptors can be a primary, secondary, or even a tertiary amine. Four representative acceptors-fluorescein methyl ester, 17-beta-estradiol, 7-mercapto-4-methylcoumarin, and 6-benzylaminopurine-were chosen as a candidate acceptor for O-, S-, and N-glucosidation, respectively. These enzymatic products were purified and the structures were confirmed with mass and NMR spectra. As all isolated glucosides are beta-anomers, BcGT1 is confirmed to be an inverting enzyme. This study not only demonstrates the substrate promiscuity of BcGT1 but also showed the great application prospect of this enzyme in bioconversion of valuable bioactive molecules.
Importance of Saccharomyces cerevisiae Oxidosqualene-Lanosterol Cyclase Tyrosine 707 Residue for Chair-Boat Bicyclic Ring Formation and Deprotonation Reactions
(2008-11-06)
Wu, Tung-Kung
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Wang, Tsai-Ting
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Chang, Cheng-Hsiang
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Liu, Yuan-Ting
;
Shie, Wen-Shiang
A contact mapping strategy was applied to identity 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.
Ligand-Functionalization of BPEI-Coated YVO4:Bi3+,Eu3+ Nanophosphors for Tumor-Cell-Targeted Imaging Applications
(2013-11-01)
Chen, Yi-Chin
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Huang, Sheng-Cih
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Wang, Yu-Kuo
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Liu, Yuan-Ting
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Wu, Tung-Kung
In this study, surface-functionalized, branched polyethylenimine (BPEI)-modified YVO4:Bi3+,Eu3+ nanocrystals (NCs) were successfully synthesized by a simple, rapid, solvent-free hydrothermal method. The BPEI-coated YVO4:Bi3+,Eu3+ NCs with high crystallinity show broad-band excitation in the =250 to 400nm near-ultraviolet (NUV) region and exhibit a sharp-line emission band centered at =619nm under excitation at =350nm. The surface amino groups contributed by the capping agent, BPEI, not only improve the dispersibility and water/buffer stability of the BPEI-coated YVO4:Bi3+,Eu3+ NCs, but also provide a capability for specifically targeted biomolecule conjugation. Folic acid (FA) and epidermal growth factor (EGF) were further attached to the BPEI-coated YVO4:Bi3+,Eu3+ NCs and exhibited effective positioning of fluorescent NCs toward the targeted folate receptor overexpressed in HeLa cells or EGFR overexpressed in A431 cells with low cytotoxicity. These results demonstrate that the ligand-functionalized, BPEI-coated YVO4:Bi3+, Eu3+ NCs show great potential as a new-generation biological luminescent bioprobe for bioimaging applications. Moreover, the unique luminescence properties of BPEI-coated YVO4:Bi3+,Eu3+ NCs show potential to combine with a UVA photosensitizing drug to produce both detective and therapeutic effects for human skin cancer therapy.
Mutation of isoleucine 705 of the oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae affects lanosterol's C/D-ring cyclization and 17 alpha/beta-exocyclic side chain stereochemistry
(2011)
Wu, Tung-Kung
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Chang, Yi-Chun
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Liu, Yuan-Ting
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Chang, Cheng-Hsiang
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Wen, Hao-Yu
Site-saturated substitution in Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase at Ile705 position produced three chair-boat-chair (C-B-C) truncated tricyclic compounds, two 17 alpha-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 17 alpha/beta-exocyclic side chain stereochemistry.
ORGN 608-Enzymatic formation of multiple triterpenes by mutation of histidine 234 of the oxidosqualene-lanosterol cyclase from saccharomyces cerevisiae
(2006-09-10)
Liu, Yuan-Ting
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Wu, Tung-Kung
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生物科技學系
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Department of Biological Science and Technology
ORGN 609-Site-saturated mutagenesis of phenylalanine 445 of saccharomyces cerevisiae oxidosqualene-lanosterol cyclase results in production of isomalabaricane triterpenoid and parkeol
(2006-09-10)
Wu, Tung-Kung
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Liu, Yuan-Ting
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Chiu, Feng-Hsuan
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生物科技學系
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Department of Biological Science and Technology
Phenylalanine 445 within oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae influences C-ring cyclization and deprotonation reactions
(2006-10-12)
Wu, Tung-Kung
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Liu, Yuan-Ting
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Chiu, Feng-Hsuan
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Chang, Cheng-Hsiang
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生物科技學系
We describe the Saccharomyces cerevisiae oxidosqualene - lanosterol cyclase Phe445 site-saturated mutants that generate truncated tricyclic and altered deprotonation product profiles. Among these mutants, only polar side-chain group substitutions genetically complemented yeast viability and produced spatially related product diversity, supporting the Johnson model that cation - pi interactions between a carbocationic intermediate and an enzyme can be replaced by an electrostatic or polar side chain to stabilize the cationic intermediate, but with product differentiation.
Protein engineering of oxidosqualene-lanosterol cyclase into triterpene monocyclase
(2013)
Chang, Cheng-Hsiang
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Wen, Hao-Yu
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Shie, Wen-Shiang
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Lu, Ching-Ting
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Li, Meng-Erh
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 pi-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.
Protein Engineering of Saccharomyces cerevisiae Oxidosqualene-Lanosterol Cyclase into Parkeol Synthase
(2012-10-19)
Liu, Yuan-Ting
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Hu, Tain-Chang
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Chang, Cheng-Hsiang
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Shie, Wen-Shiang
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Wu, Tung-Kung
A Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase mutant, ERG7(T384Y/Q450H/V4541), 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.
Protein plasticity: A single amino acid substitution in the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase generates protosta-13(17),24-dien-3 beta-ol, a rearrangement product
(2008-06-19)
Wu, Tung-Kung
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Wen, Hao-Yu
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Chang, Cheng-Hsiang
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Liu, Yuan-Ting
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應用化學系
To provide insights into the structure-function relationships of oxidosqualene-lanosterol cyclase (ERG7) from Saccharomyces cerevisiae, the Phe699 was exchanged against hydrophilic polar uncharged residues Ser, Thr, Cys, Gin, and Tyr to characterize its product profile and functional role in ERG7 activity. Among the substitutions, only the ERG7(F699T) mutant produced novel protosta-13(17),24-dien-3 beta-ol as the sole truncated rearrangement product. The results suggest that Phe699Thr mutation is likely to affect the C-17 cation stabilization during the rearrangement process.
Saccharomyces cerevisiae Oxidosqualene-Lanosterol Cyclase: A Chemistry-Biology Interdisciplinary Study of the Protein's Structure-Function-Reaction Mechanism Relationships
(2008)
Wu, Tung-Kung
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Chang, Cheng-Hsiang
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Liu, Yuan-Ting
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Wang, Tsai-Ting
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生物科技學系
The oxidosqualene cyclases (EC 5.4.99-) constitute a family of enzymes that catalyze diverse cyclization/rearrangement reactions of (3S)-2,3-oxidosqualene into a distinct array of sterols and triterpenes. The relationship between the cyclization mechanism and the enzymatic structure is extremely complex and compelling. This review covers the historical achievements of biomimetic studies and current progress in structural biology, molecular genetics, and bioinformatics studies to elucidate the mechanistic and structure-function relationships of the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase-catalyzed cyclization/rearrangement reaction. (C) 2008 The Japan Chemical journal Forum and Wiley Periodicals, Inc. Chem Rec 8: 302-325; 2008: Published online in Wiley, InterScience (www.interscience.wiley.com) DOI 10. 1002/tcr.20157
Site-saturated mutagenesis of histidine 234 of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase demonstrates dual functions in cyclization and rearrangement reactions
(2006-05-17)
Wu, Tung-Kung
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Liu, Yuan-Ting
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Chang, Cheng-Hsiang
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Yu, Mei-Ting
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Wang, Hisng-Ju
Site-saturated mutagenesis experiments were carried out on the His234 residue of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7) to characterize its functional role in ERG7 activity and to determine its effect on the oxidosqualene cyclization/ rearrangement reaction. Two novel intermediates, (13 alpha H)-isomalabarica-14(26), 17E,21-trien-3 beta-ol and protosta-20,24-dien-3 beta-ol, isolated from ERG7(H234X) mutants, provided direct mechanistic evidence for formation of the chair-boat 6-6-5 tricyclic Markovnikov cation and protosteryl cation that were assigned provisionally to the ERG7-catalyzed biosynthetic pathway. In addition, we obtained mutants that showed a complete change in product specificity from lanosterol formation to either protosta-12,24-dien-3 beta-ol or parkeol production. Finally, the repeated observation of multiple abortive and/or alternative cyclization/ arrangement products from various ERG7(H234X) mutants demonstrated the catalytic plasticity of the enzyme. Specifically, subtle changes in the active site affect both the stability of the cation-pi interaction and generate product diversity.
The cysteine 703 to isoleucine or histidine mutation of the oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae generates an iridal-type triterpenoid
(2012-11-01)
Chang, Cheng-Hsiang
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Chen, Yi-Chi
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Tseng, Sheng-Wei
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Liu, Yuan-Ting
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Wen, Hao-Yu
The Cys703 to Ile or His mutation within Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase ERG7 (ERG7(C7031/H)) generates an unusual truncated bicyclic rearranged intermediate, (8R,9R,10R)-polypoda-5,13E,17E,21-tetraen-3 beta-ol, related to iridal-skeleton triterpenoid. Numerous oxidosqualene-cyclized truncated intermediates, including tricyclic, unrearranged tetracyclic with 17 alpha/beta exocyclic hydrocarbon side chain, rearranged tetracyclic, and chair chair chair tricyclic intermediates (compounds 3-9), were also isolated from the ERG7(C703X) site-saturated mutations or the ERG7(F699T/C7031) double mutation, indicating the functional role of the Cys703 residue in stabilizing the bicyclic C-8 cation and the rearranged intermediate or interacting with Phe699, and opened a new avenue of engineering ERG7 for producing biological active agents. (C) 2012 Elsevier Masson SAS. All rights reserved.
Tryptophan 587 within oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae influences substrate binding
(2009-08-16)
Liu, Yuan-Ting
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Shie, Wen-Shiang
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Wu, Tung-Kung
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生物科技學系
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Department of Biological Science and Technology