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

Fang, Jou-Yin and Chiang, Yuan-Lan and Hsieh, Yin-Cheng and Wang, Vincent C.-C. and Huang, Yen-Chieh and Chuankhayan, Phimonphan and Yang, Ming-Chi and Liu, Ming-Yih and Chan, Sunney I. and Chen, Chun-Jung (2011) Crystallization of Adenylylsulfate Reductase from Desulfovibrio gigas: A Strategy Based on Controlled Protein Oligomerization. Crystal Growth and Design, 11 (6). pp. 2127-2134. ISSN 1528-7483.

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Adenylylsulfate reductase (adenosine 5′-phosphosulfate reductase, APS reductase or APSR, E.C. 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.

Item Type:Article
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Chan, Sunney I.0000-0002-5348-2723
Chen, Chun-Jung0000-0002-5157-4288
Additional Information:© 2011 American Chemical Society. Received: October 15, 2010. Revised: April 1, 2011. Published: April 05, 2011. Published as part of the Crystal Growth & Designvirtual special issue on the 13th International Conference on the Crystallization of Biological Macromolecules (ICCBM13). We are indebted to Yuch-Cheng Jean and the supporting staff at beamlines BL13B1 and BL13C1 at the National Synchrotron Radiation Research Center (NSRRC) and Masato Yoshimura, Jeyakanthan Jeyaraman, and Hirofumi Ishii at the Taiwan contracted beamline BL12B2 at SPring-8 for technical assistance. Portions of this research were carried out at the NSRRC-NCKU Protein Crystallography Laboratory at National Cheng Kung University (NCKU). We thank Prof. Rong-Long Pan and Prof. Ping-Ling Ong for discussions and suggestions. This work was supported in part by grants from the National Science Council [NSC 95-2313-B-213-001, 95-2313-B-009-001-MY, and 98-2311-B-213-MY3] and the National Synchrotron Radiation Center [NSRRC 983RSB02 and 993RSB02] to C.-J.C.
Funding AgencyGrant Number
National Science Council (Taipei)95-2313-B-213-001
National Science Council (Taipei)95-2313-B-009-001-MY
National Science Council (Taipei)98-2311-B-213-MY3
National Synchrotron Radiation Research Center983RSB02
National Synchrotron Radiation Research Center993RSB02
Issue or Number:6
Record Number:CaltechAUTHORS:20110621-085539646
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Official Citation: Crystallization of Adenylylsulfate Reductase from Desulfovibrio gigas: A Strategy Based on Controlled Protein Oligomerization 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 Crystal Growth & Design 2011 11 (6), 2127-2134
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:24145
Deposited By: Ruth Sustaita
Deposited On:21 Jun 2011 16:31
Last Modified:09 Mar 2020 13:18

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