10 results on '"Kai-Fa Huang"'
Search Results
2. Klebsiella pneumoniae K2 capsular polysaccharide degradation by a bacteriophage depolymerase does not require trimer formation
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Ting-Juan Ye, Kit-Man Fung, I-Ming Lee, Tzu-Ping Ko, Chia-Yi Lin, Chia-Ling Wong, I-Fan Tu, Tzu-Yin Huang, Feng-Ling Yang, Yu-Pei Chang, Jin-Town Wang, Tzu-Lung Lin, Kai-Fa Huang, and Shih-Hsiung Wu
- Subjects
Klebsiella pneumonia ,bacteriophage ,tailspike protein ,O-acetylation ,intersubunit carbohydrate-binding site ,Microbiology ,QR1-502 - Abstract
ABSTRACT K2-capsular Klebsiella pneumoniae is a hypervirulent pathogen that causes fatal infections. Here, we describe a phage tailspike protein, named K2-2, that specifically depolymerizes the K2 capsular polysaccharide (CPS) of K. pneumoniae into tetrasaccharide repeating units. Nearly half of the products contained O-acetylation, which was thought crucial to the immunogenicity of CPS. The product-bound structures of this trimeric enzyme revealed intersubunit carbohydrate-binding grooves, each accommodating three tetrasaccharide units of K2 CPS. The catalytic residues and the key interactions responsible for K2 CPS recognition were identified and verified by site-directed mutagenesis. Further biophysical and functional characterization, along with the structure of a tetrameric form of K2-2, demonstrated that the formation of intersubunit catalytic center does not require trimerization, which could be nearly completely disrupted by a single-residue mutation in the C-terminal domain. Our findings regarding the assembly and catalysis of K2-2 provide cues for the development of glycoconjugate vaccines against K. pneumoniae infection.IMPORTANCEGenerating fragments of capsular polysaccharides from pathogenic bacteria with crucial antigenic determinants for vaccine development continues to pose challenges. The significance of the C-terminal region of phage tailspike protein (TSP) in relation to its folding and trimer formation remains largely unexplored. The polysaccharide depolymerase described here demonstrates the ability to depolymerize the K2 CPS of K. pneumoniae into tetrasaccharide fragments while retaining the vital O-acetylation modification crucial for immunogenicity. By carefully characterizing the enzyme, elucidating its three-dimensional structures, conducting site-directed mutagenesis, and assessing the antimicrobial efficacy of the mutant enzymes against K2 K. pneumoniae, we offer valuable insights into the mechanism by which this enzyme recognizes and depolymerizes the K2 CPS. Our findings, particularly the discovery that trimer formation is not required for depolymerizing activity, challenge the current understanding of trimer-dependent TSP activity and highlight the catalytic mechanism of the TSP with an intersubunit catalytic center.
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- 2024
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3. Integrated omics approach to unveil antifungal bacterial polyynes as acetyl-CoA acetyltransferase inhibitors
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Ching-Chih Lin, Sin Yong Hoo, Li-Ting Ma, Chih Lin, Kai-Fa Huang, Ying-Ning Ho, Chi-Hui Sun, Han-Jung Lee, Pi-Yu Chen, Lin-Jie Shu, Bo-Wei Wang, Wei-Chen Hsu, Tzu-Ping Ko, and Yu-Liang Yang
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Biology (General) ,QH301-705.5 - Abstract
In a multi-omics analysis, bacterial polyynes are found to act as antifungal agents by inhibiting the Candida albicans polyyne resistance gene ERG10, the homolog of MasL encoding acetyl-CoA acetyltransferase.
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- 2022
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4. Structural and biological insights into Klebsiella pneumoniae surface polysaccharide degradation by a bacteriophage K1 lyase: implications for clinical use
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I-Fan Tu, Tzu-Lung Lin, Feng-Ling Yang, I-Ming Lee, Wei-Lin Tu, Jiahn-Haur Liao, Tzu-Ping Ko, Wen-Jin Wu, Jia-Tsrong Jan, Meng-Ru Ho, Ching-Yi Chou, Andrew H.-J. Wang, Chung-Yi Wu, Jin-Town Wang, Kai-Fa Huang, and Shih-Hsiung Wu
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Klebsiella pnuemoniae ,K1 capsular polysaccharide ,Tailspike protein ,Polysaccharide lyase ,Pyruvylation ,Acetylation ,Medicine - Abstract
Abstract Background K1 capsular polysaccharide (CPS)-associated Klebsiella pneumoniae is the primary cause of pyogenic liver abscesses (PLA) in Asia. Patients with PLA often have serious complications, ultimately leading to a mortality of ~ 5%. This K1 CPS has been reported as a promising target for development of glycoconjugate vaccines against K. pneumoniae infection. The pyruvylation and O-acetylation modifications on the K1 CPS are essential to the immune response induced by the CPS. To date, however, obtaining the fragments of K1 CPS that contain the pyruvylation and O-acetylation for generating glycoconjugate vaccines still remains a challenge. Methods We analyzed the digested CPS products with NMR spectroscopy and mass spectrometry to reveal a bacteriophage-derived polysaccharide depolymerase specific to K1 CPS. The biochemical and biophysical properties of the enzyme were characterized and its crystal structures containing bound CPS products were determined. We also performed site-directed mutagenesis, enzyme kinetic analysis, phage absorption and infectivity studies, and treatment of the K. pneumoniae-infected mice with the wild-type and mutant enzymes. Results We found a bacteriophage-derived polysaccharide lyase that depolymerizes the K1 CPS into fragments of 1–3 repeating trisaccharide units with the retention of the pyruvylation and O-acetylation, and thus the important antigenic determinants of intact K1 CPS. We also determined the 1.46-Å-resolution, product-bound crystal structure of the enzyme, revealing two distinct carbohydrate-binding sites in a trimeric β-helix architecture, which provide the first direct evidence for a second, non-catalytic, carbohydrate-binding site in bacteriophage-derived polysaccharide depolymerases. We demonstrate the tight interaction between the pyruvate moiety of K1 CPS and the enzyme in this second carbohydrate-binding site to be crucial to CPS depolymerization of the enzyme as well as phage absorption and infectivity. We also demonstrate that the enzyme is capable of protecting mice from K1 K. pneumoniae infection, even against a high challenge dose. Conclusions Our results provide insights into how the enzyme recognizes and depolymerizes the K1 CPS, and demonstrate the potential use of the protein not only as a therapeutic agent against K. pneumoniae, but also as a tool to prepare structurally-defined oligosaccharides for the generation of glycoconjugate vaccines against infections caused by this organism.
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- 2022
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5. Serial crystallography captures dynamic control of sequential electron and proton transfer events in a flavoenzyme
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Manuel Maestre-Reyna, Cheng-Han Yang, Eriko Nango, Wei-Cheng Huang, Eka Putra Gusti Ngurah Putu, Wen-Jin Wu, Po-Hsun Wang, Sophie Franz-Badur, Martin Saft, Hans-Joachim Emmerich, Hsiang-Yi Wu, Cheng-Chung Lee, Kai-Fa Huang, Yao-Kai Chang, Jiahn-Haur Liao, Jui-Hung Weng, Wael Gad, Chiung-Wen Chang, Allan H. Pang, Michihiro Sugahara, Shigeki Owada, Yuhei Hosokawa, Yasumasa Joti, Ayumi Yamashita, Rie Tanaka, Tomoyuki Tanaka, Fangjia Luo, Kensuke Tono, Kai-Cheng Hsu, Stephan Kiontke, Igor Schapiro, Roberta Spadaccini, Antoine Royant, Junpei Yamamoto, So Iwata, Lars-Oliver Essen, Yoshitaka Bessho, Ming-Daw Tsai, Academia Sinica, RIKEN - Institute of Physical and Chemical Research [Japon] (RIKEN), Philipps Universität Marburg = Philipps University of Marburg, Thin Film Technology Center, National Central University [Taiwan] (NCU), Japan Synchrotron Radiation Research Institute [Hyogo] (JASRI), Graduate School of Engineering Science [Toyonaka, Osaka], Osaka University, Taipei Medical University, The Fritz Haber Research Center for Molecular Dynamics [Jerusalem], The Hebrew University of Jerusalem (HUJ), Università degli Studi del Sannio, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), European Synchroton Radiation Facility [Grenoble] (ESRF), RIKEN SPring-8 Center [Hyogo] (RIKEN RSC), National Taiwan University [Taiwan] (NTU), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)
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MESH: Oxidation-Reduction ,MESH: Electrons ,Crystallography ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,General Chemical Engineering ,MESH: Crystallography ,MESH: Arginine ,Electrons ,General Chemistry ,Arginine ,Electron Transport ,MESH: Deoxyribodipyrimidine Photo-Lyase ,MESH: Flavin-Adenine Dinucleotide ,Flavins ,Flavin-Adenine Dinucleotide ,MESH: Protons ,Protons ,MESH: Electron Transport ,MESH: Flavins ,Deoxyribodipyrimidine Photo-Lyase ,Oxidation-Reduction - Abstract
International audience; Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FAD•- isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine-aspartate salt bridge allows proton transfer from arginine to FAD•-. Our molecular videos demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis.
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- 2022
6. Synergic action of an inserted carbohydrate-binding module in a glycoside hydrolase family 5 endoglucanase
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Ting-Juan Ye, Kai-Fa Huang, Tzu-Ping Ko, and Shih-Hsiung Wu
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Cellulase ,Glycoside Hydrolases ,Structural Biology ,Polysaccharides ,Catalytic Domain ,Tryptophan ,Substrate Specificity - Abstract
Most known cellulase-associated carbohydrate-binding modules (CBMs) are attached to the N- or C-terminus of the enzyme or are expressed separately and assembled into multi-enzyme complexes (for example to form cellulosomes), rather than being an insertion into the catalytic domain. Here, by solving the crystal structure, it is shown that MtGlu5 from Meiothermus taiwanensis WR-220, a GH5-family endo-β-1,4-glucanase (EC 3.2.1.4), has a bipartite architecture consisting of a Cel5A-like catalytic domain with a (β/α)8 TIM-barrel fold and an inserted CBM29-like noncatalytic domain with a β-jelly-roll fold. Deletion of the CBM significantly reduced the catalytic efficiency of MtGlu5, as determined by isothermal titration calorimetry using inactive mutants of full-length and CBM-deleted MtGlu5 proteins. Conversely, insertion of the CBM from MtGlu5 into TmCel5A from Thermotoga maritima greatly enhanced the substrate affinity of TmCel5A. Bound sugars observed between two tryptophan side chains in the catalytic domains of active full-length and CBM-deleted MtGlu5 suggest an important stacking force. The synergistic action of the catalytic domain and CBM of MtGlu5 in binding to single-chain polysaccharides was visualized by substrate modeling, in which additional surface tryptophan residues were identified in a cross-domain groove. Subsequent site-specific mutagenesis results confirmed the pivotal role of several other tryptophan residues from both domains of MtGlu5 in substrate binding. These findings reveal a way to incorporate a CBM into the catalytic domain of an existing enzyme to make a robust cellulase.
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- 2022
7. Structural and biological insights into Klebsiella pneumoniae surface polysaccharide degradation by a bacteriophage K1 lyase: implications for clinical use
- Author
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I-Fan Tu, Tzu-Lung Lin, Feng-Ling Yang, I-Ming Lee, Wei-Lin Tu, Jiahn-Haur Liao, Tzu-Ping Ko, Wen-Jin Wu, Jia-Tsrong Jan, Meng-Ru Ho, Ching-Yi Chou, Andrew H.-J. Wang, Chung-Yi Wu, Jin-Town Wang, Kai-Fa Huang, and Shih-Hsiung Wu
- Subjects
Klebsiella pnuemoniae ,K1 capsular polysaccharide ,Endocrinology, Diabetes and Metabolism ,Biochemistry (medical) ,Clinical Biochemistry ,Lyases ,Acetylation ,Cell Biology ,General Medicine ,Klebsiella Infections ,Kinetics ,Klebsiella pneumoniae ,Mice ,Polysaccharide lyase ,Medicine ,Animals ,Humans ,Pharmacology (medical) ,Bacteriophages ,Molecular Biology ,Tailspike protein ,Pyruvylation ,Bacterial Capsules - Abstract
Background K1 capsular polysaccharide (CPS)-associated Klebsiella pneumoniae is the primary cause of pyogenic liver abscesses (PLA) in Asia. Patients with PLA often have serious complications, ultimately leading to a mortality of ~ 5%. This K1 CPS has been reported as a promising target for development of glycoconjugate vaccines against K. pneumoniae infection. The pyruvylation and O-acetylation modifications on the K1 CPS are essential to the immune response induced by the CPS. To date, however, obtaining the fragments of K1 CPS that contain the pyruvylation and O-acetylation for generating glycoconjugate vaccines still remains a challenge. Methods We analyzed the digested CPS products with NMR spectroscopy and mass spectrometry to reveal a bacteriophage-derived polysaccharide depolymerase specific to K1 CPS. The biochemical and biophysical properties of the enzyme were characterized and its crystal structures containing bound CPS products were determined. We also performed site-directed mutagenesis, enzyme kinetic analysis, phage absorption and infectivity studies, and treatment of the K. pneumoniae-infected mice with the wild-type and mutant enzymes. Results We found a bacteriophage-derived polysaccharide lyase that depolymerizes the K1 CPS into fragments of 1–3 repeating trisaccharide units with the retention of the pyruvylation and O-acetylation, and thus the important antigenic determinants of intact K1 CPS. We also determined the 1.46-Å-resolution, product-bound crystal structure of the enzyme, revealing two distinct carbohydrate-binding sites in a trimeric β-helix architecture, which provide the first direct evidence for a second, non-catalytic, carbohydrate-binding site in bacteriophage-derived polysaccharide depolymerases. We demonstrate the tight interaction between the pyruvate moiety of K1 CPS and the enzyme in this second carbohydrate-binding site to be crucial to CPS depolymerization of the enzyme as well as phage absorption and infectivity. We also demonstrate that the enzyme is capable of protecting mice from K1 K. pneumoniae infection, even against a high challenge dose. Conclusions Our results provide insights into how the enzyme recognizes and depolymerizes the K1 CPS, and demonstrate the potential use of the protein not only as a therapeutic agent against K. pneumoniae, but also as a tool to prepare structurally-defined oligosaccharides for the generation of glycoconjugate vaccines against infections caused by this organism.
- Published
- 2021
8. Processive cleavage of substrate at individual proteolytic active sites of the Lon protease complex
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Kai-Fa Huang, Shih-Chieh Su, Shanshan Li, Chung-I Chang, Chiao-I Kuo, Kaiming Zhang, and Kan-Yen Hsieh
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Adenosine triphosphatase ,Multidisciplinary ,Biochemistry ,Structural Biology ,Chemistry ,Lon Protease ,SciAdv r-articles ,bacteria ,Substrate (chemistry) ,Biomedicine and Life Sciences ,Cleavage (embryo) ,Substrate degradation ,Research Article - Abstract
Description, One-way translocation and processive cleavage of substrate polypeptide occur in each of the Lon proteolytic active sites., The Lon protease is the prototype of a family of proteolytic machines with adenosine triphosphatase modules built into a substrate degradation chamber. Lon is known to degrade protein substrates in a processive fashion, cutting a protein chain processively into small peptides before commencing cleavages of another protein chain. Here, we present structural and biochemical evidence demonstrating that processive substrate degradation occurs at each of the six proteolytic active sites of Lon, which forms a deep groove that partially encloses the substrate polypeptide chain by accommodating only the unprimed residues and permits processive cleavage in the C-to-N direction. We identify a universally conserved acidic residue at the exit side of the binding groove indispensable for the proteolytic activity. This noncatalytic residue likely promotes processive proteolysis by carboxyl-carboxylate interactions with cleaved intermediates. Together, these results uncover a previously unrecognized mechanism for processive substrate degradation by the Lon protease.
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- 2021
9. Structure, dynamics, and stability of the smallest and most complex 71 protein knot.
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Min-Feng Hsu, Sriramoju, Manoj Kumar, Chih-Hsuan Lai, Yun-Ru Chen, Jing-Siou Huang, Tzu-Ping Ko, Kai-Fa Huang, and Shang-Te Danny Hsu
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MOLECULAR size , *KNOT theory , *CHEMICAL stability , *X-ray crystallography , *PROTEINS , *NUCLEAR magnetic resonance spectroscopy , *FLUORIMETRY - Abstract
Proteins can spontaneously tie a variety of intricate topological knots through twisting and threading of the polypeptide chains. Recently developed artificial intelligence algorithms have predicted several new classes of topological knotted proteins, but the predictions remain to be authenticated experimentally. Here, we showed by X-ray crystallography and solution-state NMR spectroscopy that Q9PR55, an 89-residue protein from Ureaplasma urealyticum, possesses a novel 71 knotted topology that is accurately predicted by AlphaFold 2, except for the flexible N terminus. Q9PR55 is monomeric in solution, making it the smallest and most complex knotted protein known to date. In addition to its exceptional chemical stability against urea-induced unfolding, Q9PR55 is remarkably robust to resist the mechanical unfolding-coupled proteolysis by a bacterial proteasome, ClpXP. Our results suggest that the mechanical resistance against pulling-induced unfolding is determined by the complexity of the knotted topology rather than the size of the molecule. [ABSTRACT FROM AUTHOR]
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- 2024
- Full Text
- View/download PDF
10. Processive cleavage of substrate at individual proteolytic active sites of the Lon protease complex.
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Shanshan Li, Kan-Yen Hsieh, Chiao-I Kuo, Shih-Chieh Su, Kai-Fa Huang, Kaiming Zhang, and Chung-I Chang
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TRYPSIN , *POLYACRYLAMIDE gel electrophoresis , *MOLECULAR motor proteins , *PHYSICAL sciences , *LYSOZYMES , *RECOMBINANT proteins , *RIBOSOMAL proteins , *LIFE sciences - Abstract
The article focuses on processive cleavage of substrate at individual proteolytic active sites of the Lon protease complex. Topics include the Lon protease is the prototype of a family of proteolytic machines with adenosine triphosphatase modules built into a substrate degradation chamber, and the Lon is known to degrade protein substrates in a processive fashion, cutting a protein chain processively into small peptides before commencing cleavages of another protein chain.
- Published
- 2021
- Full Text
- View/download PDF
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