1. Crystallization of Adenylylsulfate Reductase from Desulfovibrio gigas: A Strategy Based on Controlled Protein Oligomerization
- Author
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Chun-Jung Chen, Vincent C.-C. Wang, Yen-Chieh Huang, Phimonphan Chuankhayan, Sunney I. Chan, Ming-Yih Liu, Ming-Chi Yang, Yuan-Lan Chiang, Yin-Cheng Hsieh, and Jou-Yin Fang
- Subjects
chemistry.chemical_classification ,Adenosine monophosphate ,Stereochemistry ,General Chemistry ,Reductase ,Random hexamer ,Condensed Matter Physics ,Amino acid ,chemistry.chemical_compound ,Enzyme ,chemistry ,Biochemistry ,Dissimilatory sulfate reduction ,Desulfovibrio gigas ,Protein oligomerization ,General Materials Science - Abstract
Adenylylsulfate reductase (adenosine 5′-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–α_2β_2, tetramers–α_4β_4, hexamers–α_6β_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′-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 β-mercaptoethanol decreased the enzyme size from a hexamer to a dimer, probably by disrupting the disulfide Cys156—Cys162 toward the C-terminus of the β-subunit. Alignment of the APSR sequences from D. gigas and A. fulgidus revealed the largest differences in this region of the β-subunit, with the D. gigas APSR containing 16 additional amino acids with the Cys156—Cys162 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.
- Published
- 2011