Crystallization of Adenylylsulfate Reductase from Desulfovibrio gigas: A Strategy Based on Controlled Protein Oligomerization
| Autor: | 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, Jou-Yin Fang |
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| Rok vydání: | 2011 |
| Předmět: |
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 |
| Zdroj: | Crystal Growth & Design. 11:2127-2134 |
| ISSN: | 1528-7505 1528-7483 |
| DOI: | 10.1021/cg1013818 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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