Crystallization of Adenylylsulfate Reductase from Desulfovibrio gigas: A Strategy Based on Controlled Protein Oligomerization

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• 作者：Jou-Yin Fang ; Yuan-Lan Chiang ; Yin-Cheng Hsieh ; Vincent C.-C. Wang ; Yen-Chieh Huang ; Phimonphan Chuankhayan ; Ming-Chi Yang ; Ming-Yih Liu ; Sunney I. Chan ; Chun-Jung Chen
• 刊名：Crystal Growth & Design
• 出版年：2011
• 出版时间：June 1, 2011
• 年：2011
• 卷：11
• 期：6
• 页码：2127-2134
• 全文大小：997K
• 年卷期：v.11,no.6(June 1, 2011)
• ISSN：1528-7505

文摘

Adenylylsulfate reductase (adenosine 5鈥?phosphosulfate reductase, APS reductase or APSR, E.C.1.8.99.2) catalyzes the conversion of APS to sulfite in dissimilatory sulfate reduction. APSR was isolated and purified directly from massive anaerobically grown Desulfovibrio gigas, a strict anaerobe, for structure and function investigation. Oligomerization of APSR to form dimers鈥撐?sub>2尾₂, tetramers鈥撐?sub>4尾₄, hexamers鈥撐?sub>6尾₆, and larger oligomers was observed during purification of the protein. Dynamic light scattering and ultracentrifugation revealed that the addition of adenosine monophosphate (AMP) or adenosine 5鈥?phosphosulfate (APS) disrupts the oligomerization, indicating that AMP or APS binding to the APSR dissociates the inactive hexamers into functional dimers. Treatment of APSR with 尾-mercaptoethanol decreased the enzyme size from a hexamer to a dimer, probably by disrupting the disulfide Cys156鈥擟ys162 toward the C-terminus of the 尾-subunit. Alignment of the APSR sequences from D. gigas and A. fulgidus revealed the largest differences in this region of the 尾-subunit, with the D. gigas APSR containing 16 additional amino acids with the Cys156鈥擟ys162 disulfide. Studies in a pH gradient showed that the diameter of the APSR decreased progressively with acidic pH. To crystallize the APSR for structure determination, we optimized conditions to generate a homogeneous and stable form of APSR by combining dynamic light scattering, ultracentrifugation, and electron paramagnetic resonance methods to analyze the various oligomeric states of the enzyme in varied environments.