厭氧菌Desulfovibrio gigas的磷酸腺苷還原酵素所顯示可能的自我調控機制

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研究生：	江元郎 Yuan-Lang Chian
論文名稱：	厭氧菌Desulfovibrio gigas的磷酸腺苷還原酵素所顯示可能的自我調控機制 Crystal structure of adenylylsulfate reductase from Desulfovibrio gigas reveals a potential self-regulation mechanism
指導教授：	吳文桂 Wen-Guey Wu 陳俊榮 Chun-Jung Chen
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2008
畢業學年度：	96
語文別：	英文
論文頁數：	68
中文關鍵詞：	adenylylsulfate reductase 、APS reductase 、dissimilatory sulfate reduction
外文關鍵詞：	adenylylsulfate reductase, APS reductase, dissimilatory sulfate reduction
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Adenylylsulfate reductase (APSR or APS reductase) plays a key role to catalyze APS to sulfite at the dissimilatory sulfate reduction. The 500-kDa APS reductase is isolated and purified directly from the D. gigas for crystallization. The alignment of APSR sequence from D. gigas and A. fulgidus exhibits a largest difference at C-terminal of β subunit. The overall structure of APS reductase consists of six αβ-heterodimers to form a hexamer structure. The α subunit shows no-covalent bonds with FAD, and two [4Fe-4S] clusters are enveloped by cluster-binding motifs. The difference of reduced potentials at two clusters is due to the number of polar interactions between the clusters and the protein matrixes. The entrance of substrates-binding channel on α subunit shows larger space comparing with the structure from A. fulgidus because of the shift of the loop (A326-A332) and the α helix (A289-A299) in the α subunit. The C-terminal of β subunit wraps around α-subunit to form a functional unit, and the loop of C-terminal of β subunit inserts into the active channel of α subunit from another αβ-heterodimer. The interactions between the substrate-binding residue (Arg-282) and the residues (Asp-159) on the C-terminal of β subunit affect binding of the substrate. The dynamic light scattering experiment shows the multiple forms of APS reductase changes from hexamer to dimmer after adding AMP. From structure of APSR and the feature of polymerization, the hypothetic model is suggested that APSR may self regulate the activity by the C-terminal of β subunit blocking active site.
Keywords: adenylylsulfate reductase, APS reductase, dissimilatory sulfate reduction

Table of Contents
CHAPTER 1  INTRODUCTION    1
1.1    Sulfate-reducing bacteria (SRB)    1
1.2    Metabolisms of sulfate    2
1.3    Pathway of dissimilatory sulfate reduction in Desulfovibrio species    2
1.4    The model of catalytic mechanism of adenylylsulfate reductase    3
1.5    Researching aims in this thesis    6
CHAPTER 2  MATERIALS AND METHODS    7
2.1    Growth of organisms and preparation of crude extracts    7
2.2    The purification of APS reductase from D. gigas    7
2.3    Activity assay of APS reductase    8
2.4    Sequencing    8
2.5    Protein crystallization    9
2.5.1    Methods of protein crystallization    10
2.5.2    Crystallization of  APS reductase    10
2.6    X –ray Data collection and process    11
2.7    Structure determination and refinement    11
2.7.1    Phase problem and methods for phase problem    12
2.7.2    Model building and structure refinement    13
CHAPTER 3   RESULTS AND DISCUSSIONS    14
3.1    The purification and characterization of APS reductase    14
3.2    The sequence of APS reductse from D. gigas    15
3.3    Crystallographic Data and structure determine    16
3.4    The overall structure of APS reductase    17
3.5    Structure of α subunit and β subunit    18
3.6    Comparing the structure of APS reductase from A. fulgidus and D. gigas    21
3.7    Substrates binding channel and binding site    22
3.8    Interactions between the different αβ-heterodimers    25
CHAPTER 4  CONCLUSSIONS    28
Reference    30
Tables and Figures    33
Appendix 1 DNA sequence of β subunit    66
Appendix 2 DNA sequence of α subunit    67


List of tables
Table 1 The medium for growth of desulfuvibrio gigas    33
Table 2 Wolfe’s Min. Solution    33
Table 3 Sequence of primer    34
Table 4 PCR condition    34
Table 5 The condition of crystallization    34
Table 6 The activity assay during the process of purification    34
Table 7 Crystallographic Data and structure refinement    35
Table 8 Interactions between FAD-binding domain and FAD    36
Table 9 Interactions between cluster-binding motifs and residues    36
Table 10 Interactions between β subunit and the active channel    37



List of figures
FIGURE 1   FIGURE OF DESULFOVIBRIO GIGAS    38
FIGURE 2   PATHWAY OF ASSIMILATORY SULFATE REDUCTION IN E. COLI    38
FIGURE 3   PATHWAY OF DISSIMILATORY SULFATE REDUCTION    39
FIGURE 4   DISSIMILATORY SULFATE REDUCTION IN SRB    39
FIGURE 5   LOCUS OF GENES OF APSR IN DESILFOVIBRIO SPP.    40
FIGURE 6   MODEL FOR SULFATE REDUCTION IN D. VULGARIS    40
FIGURE 7   THE DOMAIN STRUCTURE OF APS REDUCTASE FROM A. FULGIDUS    41
FIGURE 8   SCHEMATIC REPRESENTATION OF THE TWO CLUSTERS BINDING SITE    41
FIGURE 9   THE CATALYTIC MECHANISM OF APS REDUCTASE    42
FIGURE 10  THE ELECTRON TRANSFER CHAIN IN ACTIVITY ASSAY    43
FIGURE 11  UV ABSORPTION OF ACTIVITY ASSAY    43
FIGURE 12  THE PHASE DIAGRAM OF VAPOR DIFFUSION PROCESS    44
FIGURE 13  VAPOR DIFFUSION METHOD    44
FIGURE 14  FIGURES OF PROTEIN CRYSTALS    45
FIGURE 15  SDS-PAGE OF THE PROCESS OF PURIFICATION    46
FIGURE 16  SEPARATED PEAKS FOLLOWING THROUGH MONO Q COLUMN    46
FIGURE 17  MOLECULAR WEIGHT ASSAY BY ULTRACENTRIFUGATION    47
FIGURE 18  UV SPECTRUM OF THE OXIDIZED APS REDUCTASE FROM D.GIGAS    48
FIGURE 19  UV SPECTRUMN OF THE OXIDIZED APSR FROM OTHER SPECIES    49
FIGURE 20  SEQUENCE ALIGNMENT OF Α SUBUNIT OF APS REDUCTASE    50
FIGURE 21  SEQUENCE ALIGNMENT OF Β SUBUNIT OF APS REDUCTASE    51
FIGURE 22  STRUCTURE OF APS REDUCTASE HEXAMER    52
FIGURE 23  STRUCTURE OF Α SUBUNIT OF APS REDUCTASE FROM D. GIGAS    53
FIGURE 24  STRUCTURE OF Β SUBUNIT OF APS REDUCTASE FROM D. GIGAS    53
FIGURE 25  SUPERIMPOSITION OF THE FAD-BINDING DOMAIN    54
FIGURE 26  THE ALIGNMENT OF CLUSTER-BINDING DOMAIN IN Β SUBUNIT    55
FIGURE 27  B-FACTOR LABEL STRUCTURE OF ΑΒ-HETERODIMER    55
FIGURE 28  THE ELECTRON DENSITY MAP AT THE C-TERMINAL OF Β SUBUNIT    56
FIGURE 29  SUPERIMPOSING APSR STRUCTURE FROM D. GIGAS AND A. FULGIDUS    57
FIGURE 30  THE ACTIVE SITE CHANNEL OF APS REDUCTASE    58
FIGURE 31  SUPERIMPOSING ELECTRON-TRANSFERRED RESIDUES ON Β SUBUNIT    59
FIGURE 32  SUPERIMPOSITION OF THE LOOP AT THE ENTRANCE OF CHANNEL (A.A.80-A.A.89)    60
FIGURE 33  INTERACTION BETWEEN Α SUBUNIT AND Β SUBUNIT    60
FIGURE 34  THE SHIFT OF THE LOOP AND HELIXS AT THE CHANNEL ENTRANCE    61
FIGURE 35  SUPERIMPOSITION OF THE SUBSTRATES BINDING SITE ON Α SUBUNIT    61
FIGURE 36  DIRECTIONS OF THE RESIDUE ARG-A336 IN DIFFERENT STATE    62
FIGURE 37  LOOP OF C-TERMINAL OF Β SUBUNIT BLOCKS SUBSTRATES BINDING SITE    63
FIGURE 38  B-FACTOR LABEL STRUCTURE OF HETEROTETRAMER    64
FIGURE 39  SUPERIMPOSITION OF SIX ΑΒ-HETERODIMER    64
FIGURE 40  SUPERIMPOSITION OF SIX C-TERMINAL LOOPS OF Β SUBUNIT    65
FIGURE 41  POLYMERIZATION OF APSR IS OBSERVED IN DLS EXPERIMENT    65

                                

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