CBM21之芳香環和極性殘基與澱粉結合時扮演不同的角色

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研究生：	張芳瑜 Fan-Yu Chang
論文名稱：	CBM21之芳香環和極性殘基與澱粉結合時扮演不同的角色 The aromatic and polar residues of CBM21 from Rhizopus oryzae glucoamylase play distinct roles in ligand-binding
指導教授：	張大慈 Margaret Dah-Tsyr Chang
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子與細胞生物研究所 Institute of Molecular and Cellular Biology
論文出版年：	2007
畢業學年度：	95
語文別：	英文
論文頁數：	71
中文關鍵詞：	澱粉、米根黴菌、葡糖澱粉酵素、澱粉吸附區
外文關鍵詞：	starch, CBM21, Rhizopus oryzae, glucoamylase, starch-binding domain
相關次數：	點閱：2 下載：0
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中文摘要
米根黴菌 (Rhizopus oryzae) 的葡糖澱粉酵素 (glucoamylase, GA) 為一種醣類水解酶，包含位於胺基端的澱粉吸附區 (starch-binding domain, SBD) 與羧基端的催化區。SBD隸屬於醣類吸附模組 (carbohydrate-binding module, CBM) 家族二十一，能與顆粒狀生澱粉進行親合性的結合。SBD的主要功能為促使配體與GA結合、增加催化區活化位置的配體濃度、以提升酵素水解效率。SBD與配體的結合主要受芳香環胺基酸與醣類的六角環形成交互作用，以及極性胺基酸與多醣的羧基形成氫鍵影響。根據核磁共振與結晶繞射解析出的米根黴菌葡糖澱粉酵素之澱粉結合區(RoGACBM21) 三級結構，天門冬胺酸29、酪胺酸32、離胺酸34、色胺酸47、天門冬胺酸50、苯丙胺酸58、酪胺酸67、榖胺酸68、酪胺酸83、酪胺酸93、酪胺酸94、天門冬胺酸96和天門冬胺酸101可能為參與配體結合的重要胺基酸。本研究以單點突變、旋光儀分析蛋白質二級結構，以及定量結合試驗檢視此B型CBM和多醣之間的疏水性作用力與氫鍵結合力。結果顯示芳香環與極性胺基酸在結合配體時扮演不同的角色，前者主要具結合配體的功能，後者則在SBD與澱粉結合後，促使澱粉支鏈鬆弛，讓配體暴露出更多的表面積，使得更多SBD得以與配體結合。本研究的重要發現為：所有具SBD功能的CBM家族 (CBM20, CBM25, CBM26, CBM34, CBM41, CBM45, and CBM48) 均可找到與RoGACBM21參與配體結合的關鍵芳香環胺基酸相對應之分子。此結果顯示即使CBM家族之間的一級胺基酸序列相似度極低，具有結合澱粉功能之SBD二級和三級結構中重要的胺基酸分子皆能在演化過程中高度保留。

Abstract
Glucoamylase (GA) from Rhizopus oryzae, a glycoside hydrolase, consists of two functional domains, an N-terminal starch-binding domain (SBD) and a C-terminal catalytic domain. The SBD is classified as a member of the carbohydrate-binding module (CBM) family 21 and possesses a high binding affinity toward raw starch. SBD promotes interaction between the ligand and GA, and increases local ligand concentration at the active site of the catalytic domain. In terms of ligand-binding, the stacking of aromatic residues against the sugar rings of polysaccharides/oligosaccharides acts as a key determinant of overall affinity and specificity, and direct hydrogen bonding between the hydroxyl groups of carbohydrates and polar residues in the binding sites of CBMs is also characteristic. Based on the exact three dimensional structure of the SBD in the presence of a cyclic ligand β-cyclodextrin (βCD) determined by NMR and X-ray crystallography, potential ligand-binding residues (Asn29, Tyr32, Lys34, Trp47, Asn50, Phe58, Tyr67, Glu68, Tyr83, Tyr94, Asn96 and Asn101) have been suggested. Here site-directed mutagenesis, circular dichroism (CD), and quantitative binding assays were performed to characterize important hydrophobic stacking interactions and direct hydrogen bonds between our type B CBM and polysaccharides/oligosaccharides. Our results reveal that aromatic and polar residues play distinct roles in ligand-binding. The former act as the major ligand-binding moieties, whereas the latter assist in forcing starch strands twisting apart to expose more ligand surface for more SBD binding. Interestingly, all key aromatic residues characterized in our SBD have counterparts in the other SBD-containing families including CBM20, CBM25, CBM26, CBM34, CBM41, CBM45, and CBM48. Taken together, our results indicate that although only extremely low sequence homology exists among these SBD-containing CBM families, functionally important residues have been well conserved through evolution.

Table of Contents
中文摘要    I
Abstract...............................................................................................................................II
Acknowledgement    III
List of Figures    VI
List of Tables    VII
Abbreviations    VIII


Chapter 1  Introduction    1
Chapter 2  Materials and Methods    4
2.1 Microorganisms and plasmids    4
2.2 Construction of plasmid    4
2.3 Site-directed mutagenesis    5
2.4 Preparation of competent cells    5
2.5 Transformation    6
2.6 Mini-preparation of plasmid    6
2.7 DNA sequencing    7
2.8 Small scale expression of RoGACBM21 derivatives    7
2.9 Large scale expression of recombinant protein    7
2.10 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)    8
2.11 Western blotting analysis    8
2.12 Determination of protein concentration    9
2.13 Purification of RoGACBM21 derivatives by amylose resin chromatography    9
2.14 Circular Dichroism spectrometry    10
2.15 Quantitative measurement of binding to granular corn starch    10
2.16 Quantitative measurement of binding to soluble polysaccharides    11
2.17 Structure-based multiple sequence alignment    12
2.18 Model construction    12
Chapter 3  Results    13
3.1 Construction of RoGACBM21 mutants    13
3.2 Expression and purification of recombinant RoGACBM21    14
3.3 Molecular weight determination of recombinant RoGACBM21    15
3.4 Secondary structure analysis of RoGACBM21    16
3.5 Starch-binding affinity and capacity    17
3.5.1 Wild-type RoGACBM21    17
3.5.2 RoGACBM21 mutant with point mutation of aromatic residue    18
3.5.3 Wild-type RoGACBM21 with fusion tag    18
3.5.4 RoGACBM21 mutant with point mutation of polar residue    19
3.6 Quantitative characterization of β-cyclodextrin binding by RoGACBM21    19
3.6.1 Wild-type RoGACBM21    20
3.6.2 RoGACBM21 mutant with point mutation of aromatic residue    20
3.6.3 RoGACBM21 mutant with point mutation of polar residue    20
3.7 A conserved mode of starch recognition    21
3.7.1 Conservation of binding residues in CBM21 members    21
3.7.2 Conservation of binding residues in starch-binding CBM families    22
3.7.3 Prediction of putative binding residues in CBM45 in the absence structure information    24
References    30
Appendix    69

List of Figures

Figure 1  Computer modeling of Rhizopus oryzae glucoamylase    35
Figure 2  Typical architectures of starch binding CBMs (SBDs)    36
Figure 3  Solution structure of RoGACBM21    37
Figure 4  Overexpression and purification of wild-type and aromatic mutant forms of RoGACBM21 in E. coli    38
Figure 5  Overexpression and purification of wild-type and polar mutant forms of RoGACBM21-6□His in E. coli    39
Figure 6  SDS-PAGE and Western blotting of RoGACBM21    40
Figure 7  Determination of the secondary structures of wild-type and aromatic mutant forms of RoGACBM21 by far-UV CD    42
Figure 8  Determination of the secondary structures of wild-type and polar mutant forms of RoGACBM21-6□His by far-UV CD    43
Figure 9  Binding of wild-type and aromatic mutant forms of RoGACBM21 to granular corn starch    44
Figure 10  Binding of wild-type and polar mutant forms of RoGACBM21-6□His to granular corn starch    46
Figure 11  Binding of RoGACBM21 to βCD    48
Figure 12  Sequence alignment of CBM21 members    49
Figure 13  Structure features of SBD from CBM21 members    50
Figure 14  Structure-based multiple sequence alignment of SBD from the individual CBM families    51
Figure 15  The structure features of SBD from individual CBM families    52
Figure 16  Molecular modeling of CBM45 using structure-based sequence alignment    53
Figure 17  Localization of residues potentially involved in maintaining structure stability    54
Figure 18  Localization of residues potentially involved in maintaining secondary structure    55
Figure 19  Localization of residues potentially involved in binding to branched chain of starch    56

List of Tables

Table 1  Starch-binding CBM families[14]    57
Table 2  Oligonucleotide primers for site-directed mutagenesis    58
Table 3  Purification efficiency and yield using amylose resin    59
Table 4  Apparant molecular weight of wild-type and aromatic mutant forms of RoGACBM21    60
Table 5  Apparant molecular weight of wild-type and polar mutant forms of RoGACBM21-6□His    61
Table 6  Levels (%) of secondary structure elements    62
Table 7  Binding parameters of wild-type and aromatic mutant forms of RoGACBM21 for granular corn starch determined by depletion isotherms    63
Table 8  Binding parameters of wild-type and polar mutant forms of RoGACBM21-6□His for granular corn starch determined by depletion isotherms    64
Table 9  Affinity of wild-type and aromatic mutant forms of RoGACBM21 for β-cyclodextrin determined by fluorescence titration spectra    65
Table 10  Affinity of wild-type and polar mutant forms of RoGACBM21-6□His for β-cyclodextrin determined by fluorescence titration spectra    66
Table 11  List of the residues proposed to involve in binding to βCD by molecular modeling NMR, X-ray crystallography, and binding assay    67
Table 12  Sequence identity and similarity of starch-binding CBMs    68

                                

References

1 Coutinho, P. M. and Reilly, P. J. (1997) Glucoamylase structural, functional, and evolutionary relationships. Proteins 29, 334-347
2 Sauer, J., Sigurskjold, B. W., Christensen, U., Frandsen, T. P., Mirgorodskaya, E., Harrison, M., Roepstorff, P. and Svensson, B. (2000) Glucoamylase: structure/function relationships, and protein engineering. Biochim Biophys Acta 1543, 275-293
3 Sorimachi, K., Le Gal-Coeffet, M. F., Williamson, G., Archer, D. B. and Williamson, M. P. (1997) Solution structure of the granular starch binding domain of Aspergillus niger glucoamylase bound to beta-cyclodextrin. Structure 5, 647-661
4 Henrissat, B. (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280 ( Pt 2), 309-316
5 Ashikari, T., Nakamura, N., Tanaka, Y., Kiuchi, N., Shibano, Y., Tanaka, T., Amachi, T. andYoshizumi, H. (1986) Rhizopus raw-starch-degrading glucoamylase: its cloning andexpression in yeast. Agric. Biol. Chem. 50, 957-964
6 Bui, D. M., Kunze, I., F¨orster, S., Wartmann, T., Horstmann, C., Manteuffel, R. and and Kunze, G. (1996) Cloning and expression of an Arxula adeninivorans glucoamylase gene in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 44, 610-619
7 Houghton-Larsen, J. and Pedersen, P. A. (2003) Cloning and characterisation of a glucoamylase gene (GlaM) from the dimorphic zygomycete Mucor circinelloides. Appl Microbiol Biotechnol 62, 210-217
8 Coutinho, P. M. a. H., B. (1999) The modular structure of cellulases and other carbohydrate-active enzymes: an integrated database approach. In Genetics, Biochemistry and Ecology of Cellulose Degradation(Ohmiya, K., Hayashi, K., Sakka, K., Kobayashi, Y., Karita, S. and Kimura, T., eds), pp. 15-23. Uni Publishers Co., Tokyo
9 Hall, J. B., G. W. Ferreira, L. M. Millward-Sadler, S. J. Ali, B. R. Hazlewood, G. P. Gilbert, H. J. (1995) The non-catalytic cellulose-binding domain of a novel cellulase from Pseudomonas fluorescens subsp. cellulosa is important for the efficient hydrolysis of Avicel. Biochem J 309, 749-756
10 Southall, S. M., Simpson, P. J., Gilbert, H. J., Williamson, G. and Williamson, M. P. (1999) The starch-binding domain from glucoamylase disrupts the structure of starch. FEBS Lett 447, 58-60
11 Hayashida S, N. K., Kanlayakrit W, Teramoto Y (1989) Characteristics and function of raw-starch-affinity site on Aspergillus awamori var. kawachi glucoamylase I molecule. Agric. Biol. Chem. 53, 143-149
12 Charnock, S. J., Bolam, D. N., Turkenburg, J. P., Gilbert, H. J., Ferreira, L. M., Davies, G. J. and Fontes, C. M. (2000) The X6 "thermostabilizing" domains of xylanases are carbohydrate-binding modules: structure and biochemistry of the Clostridium thermocellum X6b domain. Biochemistry 39, 5013-5021
13 Ali, M. K., Hayashi, H., Karita, S., Goto, M., Kimura, T., Sakka, K. and Ohmiya, K. (2001) Importance of the carbohydrate-binding module of Clostridium stercorarium Xyn10B to xylan hydrolysis. Biosci Biotechnol Biochem 65, 41-47
14 Rodriguez-Sanoja, R., Oviedo, N. and Sanchez, S. (2005) Microbial starch-binding domain. Curr Opin Microbiol 8, 260-267
15 Chou, W. I., Pai, T. W., Liu, S. H., Hsiung, B. K. and Chang, M. D. (2006) The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding. Biochem J 396, 469-477
16 Liu, Y. N., Lai, Y. T., Chou, W. I., Chang, M. D. and Lyu, P. C. (2006) Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase. Biochem J
17 Bork, P., Dandekar, T., Eisenhaber, F. and Huynen, M. (1998) Characterization of targeting domains by sequence analysis: glycogen-binding domains in protein phosphatases. J Mol Med 76, 77-79
18 Coutinho, P. M. a. H., B. (1999) Carbohydrate-active enzymes: an integrated database approach. In Recent Advances in Carbohydrate Bioengineering (Gilbert, H. J., Davies, G., Henrissat, B. and Svensson, B., eds), pp. 3–12,. The Royal Society of Chemistry, Cambridge
19 Machovic, M., Svensson, B., MacGregor, E. A. and Janecek, S. (2005) A new clan of CBM families based on bioinformatics of starch-binding domains from families CBM20 and CBM21. Febs J 272, 5497-5513
20 Kelley, L. A., MacCallum, R. M. and Sternberg, M. J. (2000) Enhanced genome annotation using structural profiles in the program 3D-PSSM. J Mol Biol 299, 499-520
21 Boraston, A. B., Bolam, D. N., Gilbert, H. J. and Davies, G. J. (2004) Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem J 382, 769-781
22 Guan, L., Hu, Y. and Kaback, H. R. (2003) Aromatic stacking in the sugar binding site of the lactose permease. Biochemistry 42, 1377-1382
23 Ponyi, T., Szabo, L., Nagy, T., Orosz, L., Simpson, P. J., Williamson, M. P. and Gilbert, H. J. (2000) Trp22, Trp24, and Tyr8 play a pivotal role in the binding of the family 10 cellulose-binding module from Pseudomonas xylanase A to insoluble ligands. Biochemistry 39, 985-991
24 Pell, G., Williamson, M. P., Walters, C., Du, H., Gilbert, H. J. and Bolam, D. N. (2003) Importance of hydrophobic and polar residues in ligand binding in the family 15 carbohydrate-binding module from Cellvibrio japonicus Xyn10C. Biochemistry 42, 9316-9323
25 Xie, H., Bolam, D. N., Nagy, T., Szabo, L., Cooper, A., Simpson, P. J., Lakey, J. H., Williamson, M. P. and Gilbert, H. J. (2001) Role of hydrogen bonding in the interaction between a xylan binding module and xylan. Biochemistry 40, 5700-5707
26 Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685
27 Lobley, A., Whitmore, L. and Wallace, B. A. (2002) DICHROWEB: an interactive website for the analysis of protein secondary structure from circular dichroism spectra. Bioinformatics 18, 211-212
28 Whitmore, L. and Wallace, B. A. (2004) DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res 32, W668-673
29 Lees, J. G., Miles, A. J., Wien, F. and Wallace, B. A. (2006) A reference database for circular dichroism spectroscopy covering fold and secondary structure space. Bioinformatics 22, 1955-1962
30 Lobley, A. a. W., B.A. (2001) DICHROWEB: A Website for the analysis of protein secondary structure from circular dichroism spectra. Biophysical J. 80, 373a
31 Compton, L. A. and Johnson, W. C., Jr. (1986) Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication. Anal Biochem 155, 155-167
32 Manavalan, P. and Johnson, W. C., Jr. (1987) Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra. Anal Biochem 167, 76-85
33 Sreerama, N. and Woody, R. W. (2000) Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287, 252-260
34 Swillens, S. (1995) Interpretation of binding curves obtained with high receptor concentrations: practical aid for computer analysis. Mol Pharmacol 47, 1197-1203
35 Cuff, J. A., Clamp, M. E., Siddiqui, A. S., Finlay, M. and Barton, G. J. (1998) JPred: a consensus secondary structure prediction server. Bioinformatics 14, 892-893
36 Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. and Higgins, D. G. (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876-4882
37 Guex, N. and Peitsch, M. C. (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714-2723
38 Schwede, T., Kopp, J., Guex, N. and Peitsch, M. C. (2003) SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res 31, 3381-3385
39 Roberge, M., Lewis, R. N., Shareck, F., Morosoli, R., Kluepfel, D., Dupont, C. and McElhaney, R. N. (2003) Differential scanning calorimetric, circular dichroism, and Fourier transform infrared spectroscopic characterization of the thermal unfolding of xylanase A from Streptomyces lividans. Proteins 50, 341-354
40 Khan, M. Y., Villanueva, G. and Newman, S. A. (1989) On the origin of the positive band in the far-ultraviolet circular dichroic spectrum of fibronectin. J Biol Chem 264, 2139-2142
41 Paldi, T., Levy, I. and Shoseyov, O. (2003) Glucoamylase starch-binding domain of Aspergillus niger B1: molecular cloning and functional characterization. Biochem J 372, 905-910
42 Jun Wu, J. L., Irene Thompson, Carey J. Oliver, Shirish Shenolikar, David L. Brautigan (1998) A conserved domain for glycogen binding in protein phosphatase-1 targeting subunits. FEBS Letters 439, 185-191
43 Penninga, D., van der Veen, B. A., Knegtel, R. M., van Hijum, S. A., Rozeboom, H. J., Kalk, K. H., Dijkstra, B. W. and Dijkhuizen, L. (1996) The raw starch binding domain of cyclodextrin glycosyltransferase from Bacillus circulans strain 251. J Biol Chem 271, 32777-32784
44 Boraston, A. B., Healey, M., Klassen, J., Ficko-Blean, E., Lammerts van Bueren, A. and Law, V. (2006) A structural and functional analysis of alpha-glucan recognition by family 25 and 26 carbohydrate-binding modules reveals a conserved mode of starch recognition. J Biol Chem 281, 587-598
45 Abe, A., Tonozuka, T., Sakano, Y. and Kamitori, S. (2004) Complex structures of Thermoactinomyces vulgaris R-47 alpha-amylase 1 with malto-oligosaccharides demonstrate the role of domain N acting as a starch-binding domain. J Mol Biol 335, 811-822
46 Mikami, B., Iwamoto, H., Malle, D., Yoon, H. J., Demirkan-Sarikaya, E., Mezaki, Y. and Katsuya, Y. (2006) Crystal structure of pullulanase: evidence for parallel binding of oligosaccharides in the active site. J Mol Biol 359, 690-707
47 Feese, M. D., Kato, Y., Tamada, T., Kato, M., Komeda, T., Miura, Y., Hirose, M., Hondo, K., Kobayashi, K. and Kuroki, R. (2000) Crystal structure of glycosyltrehalose trehalohydrolase from the hyperthermophilic archaeum Sulfolobus solfataricus. J Mol Biol 301, 451-464
48 Williamson, M. P., Le Gal-Coeffet, M. F., Sorimachi, K., Furniss, C. S., Archer, D. B. and Williamson, G. (1997) Function of conserved tryptophans in the Aspergillus niger glucoamylase 1 starch binding domain. Biochemistry 36, 7535-7539
49 Giardina, T., Gunning, A. P., Juge, N., Faulds, C. B., Furniss, C. S., Svensson, B., Morris, V. J. and Williamson, G. (2001) Both binding sites of the starch-binding domain of Aspergillus niger glucoamylase are essential for inducing a conformational change in amylose. J Mol Biol 313, 1149-1159
50 Notenboom, V., Boraston, A. B., Chiu, P., Freelove, A. C., Kilburn, D. G. and Rose, D. R. (2001) Recognition of cello-oligosaccharides by a family 17 carbohydrate-binding module: an X-ray crystallographic, thermodynamic and mutagenic study. J Mol Biol 314, 797-806
51 Xie, H., Gilbert, H. J., Charnock, S. J., Davies, G. J., Williamson, M. P., Simpson, P. J., Raghothama, S., Fontes, C. M., Dias, F. M., Ferreira, L. M. and Bolam, D. N. (2001) Clostridium thermocellum Xyn10B carbohydrate-binding module 22-2: the role of conserved amino acids in ligand binding. Biochemistry 40, 9167-9176
52 Simpson, P. J., Xie, H., Bolam, D. N., Gilbert, H. J. and Williamson, M. P. (2000) The structural basis for the ligand specificity of family 2 carbohydrate-binding modules. J Biol Chem 275, 41137-41142
53 Machovic, M. and Janecek, S. (2006) Starch-binding domains in the post-genome era. Cell Mol Life Sci 63, 2710-2724

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