Browsing by Author "Liu, Sheng-Wen"

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Characterization and identification of essential residues of the glycoside hydrolase family 64 laminaripentaose-producing--1, 3-glucanase
(2011-08-01)
Shrestha, Keshab Lal
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Liu, Sheng-Wen
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Huang, Chin-Ping
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Wu, Hsin-Mao
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Wang, Wen-Ching
Laminaripentaose-producing -1,3-glucanase (LPHase) from Streptomyces matensis DIC-108 uniquely catalyzes the hydrolysis of -1,3-glucan to release laminaripentaose as the predominant product. For studying this novel enzyme, the gene of LPHase was reconstructed with polymerase chain reaction and over-expressed in Escherichia coli. The recombinant wild-type enzyme and various mutants were further purified to 90 homogeneity on an ion-exchange chromatograph. The catalysis of the recombinant LPHase is confirmed to follow a one-step single-displacement mechanism with (1)H-NMR spectrometry. To determine the amino-acid residues essential for the catalysis, more than ten residues, including five highly conserved residuesAsp(143), Glu(154), Asp(170), Asp(376) and Asp(377), were mutated. Among the mutants, E154Q, E154G, D174N and D174G significantly lost catalytic activity. Further investigation with chemical rescue using sodium azide on E154G and D174G confirmed that Glu(154) functions as the general acid whereas Asp(170) serves as the general base in a catalytic turnover. This work is the first report that provides direct information for the identification of the essential residues of GH-64 through kinetic examination.
Expression, Purification and Characterization of Human alpha-L-Fucosidase
(2009-08-01)
Liu, Sheng-Wen
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Li, Yaw-Kuen
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應用化學系
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Department of Applied Chemistry
alpha-L-Fucosidases (EC 3.2.1.51) are exo-glycosidases. On the basis of the multi-alignment of amino acid sequence, alpha-L-fucosidases were classified into two families of glycoside hydrolases, GH-29 and GH-95. They are responsible for the removal Of L-fucosyl residues from the non-reducing end of glycoconjugates. Deficiency of alpha-L-fucosidase results in Fucosidosis due to the accumulation of fucose-containing glycolipids, glycoproteins and oligosaccharides in various tissues. Recent studies discovered that the fucosylation levels are increased on the membrane surfaces of many carcinomas, indicating the biological function of alpha-L-fucosidases may relate to this abnormal cell physiology. Although the gene of human alpha-L-fucosidase (h-fuc) was cloned, the recombinant enzyme has rarely been overexpressed as a soluble and active from. We report herein that, with carefully control on the growing condition, an active human alpha-L-fucosidases (h-Fuc) was successfully expressed in Escherichia coli for the first time. After a series steps of ion-exchange and gel-filtration chromatographic purification, the recombinant h-Fuc with 95% homogeneity was obtained. The molecular weight of the enzyme was analyzed by SDS-PAGE (similar to 50 kDa) and confirmed by ESI mass (50895 Da). The recombinant h-Fuc was stable up to 55 degrees C with incubation at pH 6.8 for 2 h; the optimum temperature for h-Fuc is approximately 55 degrees C. The enzyme was stable at pH 2.5-7.0 for 2 h; the enzyme activity decreased greatly for pH greater than 8.0 or less than 2.0. The K(m) and k(cat) values of the recombinant h-Fuc (at pH 6.8) were determined to be 0.28 mM and 17.1 s(-1), respectively. The study of pH-dependent activity showed that the recombinant enzyme exhibited optimum activity at two regions near at pH 4.5 and pH 6.5. These features of the recombinant h-Fuc are comparable to the native enzyme purified directly from human liver. Studies on the transfucosylation and common intermediate of the enzymatic reaction by NMR support that h-Fuc functions as a retaining enzyme catalyzing the hydrolysis of substrate via a two-step, double displacement mechanism.
Identification of Essential Residues of Human alpha-L-Fucosidase and Tests of Its Mechanism
(2009-01-13)
Liu, Sheng-Wen
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Chen, Chao-Sheng
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Chang, Shih-Shen
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Mong, Kwok-Kong Tony
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Lin, Chun-Hung
Fucosylated glycoconjugates have critical roles in biological processes, but a limited availability Of alpha-L-fucosidase has hampered research on this human enzyme (h-Fuc) at a molecular level. After overexpressing h-Fuc in Escherichia coli as an active form, we investigated the catalytic function of this recombinant enzyme. Based on sequence alignment and structural analysis of close homologues of h-Fuc, nine residues of glutamate and aspartate in h-Fuc were selected for mutagenic tests to determine the essential residues. Among the mutants, D225N, E289Q, and E289G lost catalytic activity significantly; their k(cat) values are 1/5700, 1/430, and 1/340, respectively, of that of the wild-type enzyme. The Bronsted plot for k(cat)/K(m) for the E289G mutant is linear with beta(1g) = -0.93, but that for k(cat) is biphasic, with beta(1g) for poor substrates being -0.88 and for activated substrates being -0.11. The small magnitude of Pig for the activated substrates may indicate that the rate-limiting step of the reaction is defucosylation, whereas the large magnitude of the latter Pig value for the poor substrates indicates that the rate-limiting step of the reaction becomes fucosylation. The kinetic outcomes support an argument that ASp(225) functions as a nucleophile and Glu(289) as a general acid/base catalyst. As further evidence, azide significantly reactivated D225G and E289G, and (1)H NMR spectral analysis confirmed the formation of beta-fucosyl azide and alpha-fucosyl azide in the azide rescues of D225G and E289G catalyses, respectively. As direct evidence to prove the function of Glu(289), an accumulation of fucosyl-enzyme intermediate was detected directly through ESI/MS analysis.
Low-Complexity ICI Suppression Methods Utilizing Cyclic Prefix for OFDM Systems in High-Mobility Fading Channels
(2014-02-01)
Ma, Chun-Ying
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Liu, Sheng-Wen
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Huang, Chia-Chi
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電機資訊學士班
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Undergraduate Honors Program of Electrical Engineering and Computer Science
This paper focuses on a simple intercarrier interference (ICI) suppression scheme that linearly combines the inter-symbol interference (ISI)-free part of a cyclic prefix (CP) and its corresponding part in an orthogonal frequency-division multiplexing (OFDM) signal to soothe the ICI effect caused by a time-varying channel. In contrast with existing heuristic combining weights in the literature for ICI suppression, we derived three sets of optimum combining weights in different aspects. Through simulations, we show that the proposed combining weights outperform other existing heuristic combining weights. In addition, a simplified implementation is introduced to reduce the computational complexity of combining. Furthermore, the proposed methods can be incorporated with other ICI mitigation methods to further improve the system performance.
Metabolic labelling of cholesteryl glucosides in Helicobacter pylori reveals how the uptake of human lipids enhances bacterial virulence
(2016-01-01)
Jan, Hau-Ming
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Chen, Yi-Chi
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Shih, Yu-Yin
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Huang, Yu-Chen
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Tu, Zhijay
Helicobacter pylori infects approximately half of the human population and is the main cause of various gastric diseases. This pathogen is auxotrophic for cholesterol, which it converts upon uptake to various cholesteryl alpha-glucoside derivatives, including cholesteryl 6'-acyl and 6'-phosphatidyl alpha-glucosides (CAGs and CPGs). Owing to a lack of sensitive analytical methods, it is not known if CAGs and CPGs play distinct physiological roles or how the acyl chain component affects function. Herein we established a metabolite-labelling method for characterising these derivatives qualitatively and quantitatively with a femtomolar detection limit. The development generated an MS/MS database of CGds, allowing for profiling of all the cholesterol-derived metabolites. The subsequent analysis led to the unprecedented information that these bacteria acquire phospholipids from the membrane of epithelial cells for CAG biosynthesis. The resulting increase in longer or/and unsaturated CAG acyl chains helps to promote lipid raft formation and thus delivery of the virulence factor CagA into the host cell, supporting the idea that the host/pathogen interplay enhances bacterial virulence. These findings demonstrate an important connection between the chain length of CAGs and the bacterial pathogenicity.
On Optimum Segment Combining Weight for ICI Self-Cancellation in OFDM Systems under Doubly Selective Fading Channels
(2012)
Ma, Chun-Ying
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Liu, Sheng-Wen
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Huang, Chia-Chi
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電信工程研究所
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Institute of Communications Engineering
ICI self-cancellation algorithms are promising techniques to mitigate the ICI effect in OFDM systems caused by high mobility. In the area of ICI self-cancellation, the calculation of optimum combining weight is still an open question. In this paper, the problem of finding optimum combining weight to minimize the ICI-plus-noise power is formulated. We then derived a non-iterative algorithm to find the optimum solution. We also compare the performance of the optimum combining weight with other existing combining weight methods.
Structural study of laminaripentaose-producing beta-1,3-glucanase from Streptomyces matensis DIC-108
(2008-01-01)
Wu, Hsin-Mao
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Hsu, Mina-Tsung
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Lai, Chun-Chieh
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Liu, Sheng-Wen
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Li, Yaw-Kuen
Structure, Mechanistic Action, and Essential Residues of a GH-64 Enzyme, Laminaripentaose-producing beta-1,3-Glucanase
(2009-09-25)
Wu, Hsin-Mao
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Liu, Sheng-Wen
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Hsu, Ming-Tsung
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Hung, Chiu-Lien
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Lai, Chun-Chieh
Laminaripentaose-producing beta-1,3-glucanase (LPHase), a member of glycoside hydrolase family 64, cleaves a long-chain polysaccharide beta-1,3-glucan into specific pentasaccharide oligomers. The crystal structure of LPHase from Streptomyces matensis DIC-108 was solved to 1.62 angstrom resolution using multiple-wavelength anomalous dispersion methods. The LPHase structure reveals a novel crescent-like fold; it consists of a barrel domain and a mixed (alpha/beta) domain, forming a wide-open groove between the two domains. The liganded crystal structure was also solved to 1.80 angstrom, showing limited conformational changes. Within the wide groove, a laminaritetraose molecule is found to sit in an electronegatively charged central region and is proximal to several conserved residues including two carboxylates (Glu(154) and Asp(170)) and four other sugar-binding residues (Thr156, Asn(158), Trp(163), and Thr(167)). Molecular modeling using a laminarihexaose as a substrate suggests roles for Glu(154) and Asp(170) as acid and base catalysts, respectively, whereas the side chains of Thr(156), Asn(158), and Trp(163) demarcate subsite +5. Site-directed mutagenesis of Glu(154) and Asp(170) confirms that both carboxylates are essential for catalysis. Together, our results suggest that LPHase uses a direct displacement mechanism involving Glu(154) and Asp(170) to cleave a beta-1,3-glucan into specific alpha-pentasaccharide oligomers.