Browsing by Author "Lai, Chun-Chieh"

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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 of native oligomeric Sprouty2 by electron microscopy and its property of electroconductivity
(2013-09-27)
Chen, Feng-Jung
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Lee, Kuan-Wei
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Lai, Chun-Chieh
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Lee, Sue-Ping
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Shen, Hsiao-Hsuian
Receptor tyrosine kinases (RTKs) regulate many cellular processes, and Sprouty2 (Spry2) is known as an important regulator of RTK signaling pathways. Therefore, it is worth investigating the properties of Spry2 in more detail. In this study, we found that Spry2 is able to self-assemble into oligomers with a high-affinity KD value of approximately 16 nM, as determined through BIAcore surface plasmon resonance analysis. The three-dimensional (3D) structure of Spry2 was resolved using an electron microscopy (EM) single-particle reconstruction approach, which revealed that Spry2 is donut-shaped with two lip-cover domains. Furthermore, the method of energy dispersive spectrum obtained through EM was analyzed to determine the elements carried by Spry2, and the results demonstrated that Spry2 is a silicon- and iron-containing protein. The silicon may contribute to the electroconductivity of Spry2, and this property exhibits a concentration-dependent feature. This study provides the first report of a silicon- and iron-containing protein, and its 3D structure may allow us (1) to study the potential mechanism through the signal transduction is controlled by switching the electronic transfer on or off and (2) to develop a new type of conductor or even semiconductor using biological or half-biological hybrid materials in the future. (C) 2013 Elsevier Inc. All rights reserved.
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.