臺灣博碩士論文加值系統

醣類結合模組 (carbohydrate-binding modules, CBM) 存在於醣類水解酵素等醣類活性酵素(carbohydrate-active enzymes) 的一段獨立區塊 (domain)，有著多種輔助醣類活性蛋白的功用。其中有一大部分主要的作用是幫助這類酵素附著到非水溶性的醣類巨分子之上。 當失去醣類結合模組時，醣類活性蛋白保有著對水溶性的醣類受質之活性，但是對非水溶性的醣類受質卻幾乎完全喪失活性。
迄今，醣類活性酵素網站 (http://afmb.cnrs-mrs.fr/CAZY) 記載著四十五個家族 (families) 不同的醣類結合模組。 這些家族主要是由序列的相似性做分類。 其中已知有七個家族的成員參與澱粉的結合，分別是醣類結合模組第二十族(CBM20)、第二十一族(CBM21)、第二十五族 (CBM25)、第二十六族(CBM26)、第三十四族(CBM34)、第四十一族 (CBM41) 以及第四十五族(CBM45)。 在本篇論文中我們利用核磁共振(nuclear magnetic resonance)的方法解出米根黴的葡糖澱粉酶(glucoamylase, 此酵素在食品工業上常用於水解澱粉)之澱粉結合區段(Starch binding domain, SBD)的水溶液結構，此澱粉結合區段屬於第二十一族醣類結合模組(RoCBM21)；而此結構為第一個第二十一族醣類結合模組的代表結構。
米根黴第二十一族醣類結合模組的結構是由八個反平行的貝他平股(b-strand)所構成的類免疫球蛋白結構； 將此結構與先前解出的澱粉結合區段的分子結構做比較，我們可以將澱粉結合區段的結構歸類成兩種摺疊拓墣圖形式(topologies) ；第一型(type I topology)包含醣類結合模組第二十族、第二十五族、第二十六族以及第四十一族 (CBM41)而第二型(type II topology)則包含醣類結合模組第二十一族(CBM21)及第三十四族(CBM34) 。 此二種摺疊拓墣圖形式大致上相近除了第一型包含著一對平行的貝他平股而第二型則完全由反平行的貝他平股所構成。
化學位移干擾(Chemical shift perturbation)及先前的研究指出，米根黴二十一族醣結合模組(RoCBM21)與麴菌二十族醣結合模組(CBM20 from Aspergillus niger glucoamylase)一樣具有兩個受質結合位(binding sites)，且二者的第二結合位(site II)胺基酸組成較為相似，米根黴二十一族醣結合模組的第一結合位則由帶有多個天門冬胺酸的環圈(loops)所包圍。 此外利用電腦模擬分子泊合(docking)將由二十六個葡萄糖所組成具有結構的澱粉分子與米根黴二十一族醣結合模組進行結合模擬。在此模擬中我們看到了澱粉結合區段與澱粉自身的結構產生作用的可能性。 米根黴二十一族醣結合模的分子結構使我們能更進一步得瞭解蛋白質與醣類的結合作用。

Carbohydrate-binding modules (CBMs) function independently to assist carbohydrate-active enzymes. Family 21 CBMs contain ~100 amino acid residues, and some members have starch-binding functions or glycogen-binding activities. We report here the first structure of a family 21 CBM from the starch binding domain (SBD) of Rhizopus oryzae glucoamylase (RoCBM21) determined by nuclear magnetic resonance spectroscopy. This CBM has a ��-sandwich fold with an immunoglobulin-like structure. Ligand-binding properties of RoCBM21 were analyzed by chemical shift perturbations and automated docking. Structural comparisons with previously reported SBDs revealed two types of topologies, type I and type II, with CBM20, CBM25, CBM26, and CBM41 belonging to the type I topology group, whereas CBM21 and CBM34 belonging type II topology. According to the chemical shift perturbations, RoCBM21 contains two ligand-binding sites—as do most SBDs. Residues in site II are similar to those found in the family 20 CBM from Aspergillus niger glucoamylase (AnCBM20). Site I, however, is enclosed by two flexible asparagine-rich loops that are unique and only found in some members of CBM21s. Additionally, docking of a large amylose molecule (comprising 26 glucose residues) into RoCBM21 highlights possible mechanism that SBDs may interact with the structured portion of starch. The structure of RoCBM21 helps us characterize the structural features of SBDs and to understand the basis of protein-carbohydrate recognitions.

Part I Solution Structure of Family 21 Carbohydrate-binding Module from Rhizopus oryzae Glucoamylase....................................................................................1
Abstract (in Chinese)................................................................................................2
Abstract (in English).................................................................................................4
Abbreviations.............................................................................................................6
Chapter One: Introduction......................................................................................7
1.1. Carbohydrates.................................................................................................7
1.2. Starch and Glycogen.......................................................................................8
1.3. Carbohydrate-active Enzymes.......................................................................9
1.4. Carbohydrate-binding Modules...................................................................10
1.5. Rhizopus oryzae..............................................................................................11
1.6. Glucoamylase.................................................................................................13
1.7. Starch-binding Domain (SBD) ....................................................................14
Tables....................................................................................................................17
Figures...................................................................................................................20
Chapter Two: Materials & methods.....................................................................29
2.1. Clone Construction.......................................................................................29
2.2. Sample Purification.......................................................................................29
2.3. NMR Spectroscopy for Structure Determination......................................30
2.4. Structure Calculation and Structural Analyses.........................................33
2.5. Chemical Shift Perturbations.......................................................................35
2.6. Docking Simulations.....................................................................................36
Sequence details about RoCBM21......................................................................38
Figures...................................................................................................................39
Chapter Three: Results...........................................................................................53
3.1. NMR Spectra and Molecular Structure......................................................53
3.2. Structural Comparisons to SBDs.................................................................56
3.3. Ligand-binding and Chemical Shift Perturbation.....................................56
Tables....................................................................................................................60
Figures...................................................................................................................66
Chapter Four: Discussions.....................................................................................83
Two topology types of SBDs................................................................................83
Aromatic residues and poly-N loops in RoCBM21...........................................84
Interpretation of C-terminal deletions of glucoamylase...................................85
Diversity of SBDs..................................................................................................86
Interactions with starch and models of AFM images.......................................87
Models and studies of other CBM21s.................................................................88
Prospects...............................................................................................................89
Figures...................................................................................................................90
Reference................................................................................................................100
Part II Characterization and structural analyses of nsLTP1 from mung bean..............................................................................................................108
Abstract (in Chinese)........................................................................................... 109
Abstract (in English) ............................................................................................110
Abbreviations.........................................................................................................111
Chapter One: Introduction .................................................................................112
Figures..................................................................................................................115
Chapter Two: Materials & methods...................................................................117
Protein Purification........................................................................................117
NMR Experiments and Assignments............................................................118
Structural Calculation and Analysis.............................................................119
Figures.................................................................................................................120
Chapter Three: Results and Discussions
Assignment of NMR Spectra and Structure Calculation of nsLTP1.........123
Comparison with homologous nsLTP1s........................................................124
Tables...................................................................................................................128
Figures.................................................................................................................131
Chapter Four: Conclusions..................................................................................137
Lipid transfer activity, cavity and disulfide bonds......................................137
Probable Role of nsLTP1 as Plant Defensin.................................................139
Reference.............................................................................................................141

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