Desulfovibrio gigas中磷酸腺苷還原酶的聚合態與自我調節機制的關係探討

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研究生：	方柔尹 Fang, Jou-Yin
論文名稱：	Desulfovibrio gigas中磷酸腺苷還原酶的聚合態與自我調節機制的關係探討 Biophysical Evidence of Oligomerization Suggests a Self-Regulation Mechanism of Adenylylsulfate Reductase from Desulfovibrio gigas
指導教授：	潘榮隆 Pan, Rong-Long 陳俊榮 Chen, Chun-Jung
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：	2010
畢業學年度：	98
語文別：	英文
論文頁數：	47
中文關鍵詞：	磷酸腺苷還原酶
外文關鍵詞：	APS reductase, Adenylylsulfate reductase, APSR, Desulfovibrio gigas, D. gigas
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Adenylyl sulfate reductase (APSR or APS reductase, E.C.1.8.99.2), which was purified from the anaerobic sulfate-reducing bacterium Desulfovibrio gigas, played a key role to catalyze APS to sulfite in the dissimilatory sulfate reduction. Reduction of ferricyanide was used as reversed reaction of APSR for determining the formation of APS from AMP and sulfite. The overall structure of APSR contains six αβ-heterodimers that comprise one α-subunit and one β-subunit to form a hexamer structure. The C-terminus of the β-subunit (B105-B152) wrapped around α-subunit formed a functional unit, and the loop of the β-subunit C-terminus (B153-B167) inserted into the active channel of the α-subunit from another αβ-heterodimer. The hypothetic mode was suggested that APSR might self regulate the activity by the β-subunit C-terminus blocking the active site because the dynamic light scattering (DLS) data showed that the average diameter of APSR decreased from 12.81 to 8.11 nm after adding AMP, corresponding the size change from the hexamer to dimer structure. The sole disulfide of the structure is positioned at the tail of C-terminus of the β-subunit (Cys 156 and Cys 16), which was the key connection to keep the shape of the loop of the β-subunit C-terminus. After treating with 2-mercaptoethanol (β-me) to break the disulfide, the average diameter of APSR decreased from 12.81 to 8.47 nm. The results suggest that the dimer form could be the functional structure.

ABSTRACT    1
CHAPTER 1  INTRODUCTION    1
1.1    SULFATE-REDUCING BACTERIA (SRB)    1
1.2    DISSIMILATORY SULFATE REDUCTION    2
1.3    MECHANISM OF ADENYLYLSULFATE REDUCTASE (APSR)    3
1.4    STRUCTURE OF APSR    3
1.5  SUBSTRATES BINDING CHANNEL AND BINDING SITE    4
CHAPTER 2  MATERIALS AND METHODS    6
2.1    GROWTH OF THE ORGANISM    6
2.2    PURIFICATION OF APSR FROM D.GIAGS    6
2.3    ACTIVITY ASSAY OF APSR    8
2.4    SDS PAGE    9
2.5    DYNAMIC LIGHT SCATTERING (DLS)    10
2.5.1    Theory of DLS    10
2.5.2    Sample Preparation of DLS    12
2.5.3    Characteristics of DLS    12
2.6    ATOMIC FORCE MICROSCOPY (AFM)    13
2.6.1    Theory of Atomic Force Microscopy    13
2.6.2    Device of Atomic Force Microscopy System    14
2.6.3    Operation Mode    15
CHAPTER 3  RESULTS AND DISCUSSIONS    16
3.1    GROWTH OF THE ORGANISM    16
3.2    THE PURIFICATION AND CHARACTERIZATION OF APSR    16
3.3    MODEL OF APSR SELF-REGULATION    17
3.4    CRYSTALLOGRAPHY    17
3.5    ULTRACENTRIFUGATION    18
3.6    IMAGE OF ATOMIC FORCE MICROSCOPE (AFM)    18
3.7    DYNAMIC LIGHT SCATTERING (DLS)    20
CHAPTER 4  CONCLUSSIONS    22
LIST OF TABLES    24
TABLE. 1 THE MEDIUM FOR GROWTH OF DESULFUVIBRIO GIGAS    24
TABLE. 2 WOLFE’S MIN. SOLUTION    24
TABLE. 3 SPECIFICATION OF NANO S    25
TABLE. 4 RECOMMENDED SAMPLE CONCENTRATIONS    25
TABLE. 5 DISPERSION LEVEL OF PARTICLES    25
TABLE. 6 THE ACTIVITY ASSAY DURING THE PROCESS OF PURIFICATION    25
TABLE. 7 THE MODIFIED CRYSTALLIZATION CONDITION    26
LIST OF FIGURES    27
FIG. 1  RESPIRATORY SULFATE REDUCTION    27
FIG. 2  BIOENERGETICS OF THE GROWTH OF DESULFOTOMACULUM ON LACTATE PLUSE SULFATE    27
FIG. 3  BIOENERGETICS OF THE GROWTH OF DESULFOVIBRIO GIGAS ON LACTATE PLUSE SULFATE    28
FIG. 4  FIGURE OF DESULFOVIBRIO GIGAS    28
FIG. 5  PATHWAY OF ASSIMILATORY SULFATE REDUCTION IN E. COLI    29
FIG. 6  ACTIVITY ASSAY OF APSR    29
FIG. 7  VISIBLE/ULTRAVIOLET SPECTRUM OF APSR    29
FIG. 8  STRUCTURE OF APSR IN D.GIGAS    30
FIG. 9  ELECTRON TRANSFER OF APSR IN D.GIGAS    31
FIG. 10  THE LOOP OF C-TERMINAL OF Β SUBUNIT BLOCKS THE SUBSTRATES-BINDING SITE    31
FIG. 11  OLIGOMER STRUCTURE OF APSR    32
FIG. 12  FIGURE OF DESULFOVIBRIO GIGAS    33
FIG. 13  MACRO-DEAE COLUMN    33
FIG. 14  SDS PAGE    33
FIG. 15  UV ABSORPTION OF ACTIVITY ASSAY    34
FIG. 16  UV SPECTRUM OF THE OXIDIZED APS REDUCTASE FROM D.GIGAS    34
FIG. 17  MONO Q    35
FIG. 18  EPR DATA OF APSR P1 AND P2    35
FIG. 19  DEVICE OF DYNAMIC LIGHT SCATTERING ZETASIZER NANO SERIES    36
FIG. 20  INTERACTIVE FORCE BETWEEN ATOMS    36
FIG. 21  DEVICE OF AFM SYSTEM    37
FIG. 22  MOLECULAR MASS ASSAY BY ULTRACENTRIFUGATION    38
FIG. 23  AFM DATA OF APSR P1 AND P2    40
FIG. 24  DLS DATA OF APSR IN D.GIGAS TREATED WITH AMP AND □ -MERCAPTOETHANOL    41
FIG. 25  DLS DATA OF APSR IN D.GIGAS TREATED WITH □ -MERCAPTOETHANOL    42
FIG. 26  DLS DATA OF APSR IN D.GIGAS TREATED WITH SULFITE    43
FIG. 27  DLS DATA OF APSR IN D.GIGAS IN DIFFERENT PH VALUE    43
FIG. 28  INITIAL SCREEN CONDITION    44
FIG. 29  MODIFIED CRYSTAL    44
REFERENCES    45

                                

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