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Title: Structural and mechanistic analysis of the arsenate respiratory reductase provides insight into environmental arsenic transformations

Abstract

Arsenate respiration by bacteria was discovered over two decades ago and is catalyzed by diverse organisms using the well-conserved Arr enzyme complex. Until now, the mechanisms underpinning this metabolism have been relatively opaque. Here, we report the structure of an Arr complex (solved by X-ray crystallography to 1.6-Å resolution), which was enabled by an improved Arr expression method in the genetically tractable arsenate respirer Shewanella sp. ANA-3. We also obtained structures bound with the substrate arsenate (1.8 Å), the product arsenite (1.8 Å), and the natural inhibitor phosphate (1.7 Å). The structures reveal a conserved active-site motif that distinguishes Arr [(R/K)GRY] from the closely related arsenite respiratory oxidase (Arx) complex (XGRGWG). Arr activity assays using methyl viologen as the electron donor and arsenate as the electron acceptor display two-site ping-pong kinetics. A Mo(V) species was detected with EPR spectroscopy, which is typical for proteins with a pyranopterin guanine dinucleotide cofactor. Arr is an extraordinarily fast enzyme that approaches the diffusion limit ( K m = 44.6 ± 1.6 μM, k cat = 9,810 ± 220 seconds −1 ), and phosphate is a competitive inhibitor of arsenate reduction ( K i = 325 ± 12 μM). These observations, combined with knowledgemore » of typical sedimentary arsenate and phosphate concentrations and known rates of arsenate desorption from minerals in the presence of phosphate, suggest that ( i ) arsenate desorption limits microbiologically induced arsenate reductive mobilization and ( ii ) phosphate enhances arsenic mobility by stimulating arsenate desorption rather than by inhibiting it at the enzymatic level.« less

Authors:
; ; ; ;
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1464330
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 115 Journal Issue: 37; Journal ID: ISSN 0027-8424
Publisher:
Proceedings of the National Academy of Sciences
Country of Publication:
United States
Language:
English

Citation Formats

Glasser, Nathaniel R., Oyala, Paul H., Osborne, Thomas H., Santini, Joanne M., and Newman, Dianne K. Structural and mechanistic analysis of the arsenate respiratory reductase provides insight into environmental arsenic transformations. United States: N. p., 2018. Web. doi:10.1073/pnas.1807984115.
Glasser, Nathaniel R., Oyala, Paul H., Osborne, Thomas H., Santini, Joanne M., & Newman, Dianne K. Structural and mechanistic analysis of the arsenate respiratory reductase provides insight into environmental arsenic transformations. United States. doi:10.1073/pnas.1807984115.
Glasser, Nathaniel R., Oyala, Paul H., Osborne, Thomas H., Santini, Joanne M., and Newman, Dianne K. Mon . "Structural and mechanistic analysis of the arsenate respiratory reductase provides insight into environmental arsenic transformations". United States. doi:10.1073/pnas.1807984115.
@article{osti_1464330,
title = {Structural and mechanistic analysis of the arsenate respiratory reductase provides insight into environmental arsenic transformations},
author = {Glasser, Nathaniel R. and Oyala, Paul H. and Osborne, Thomas H. and Santini, Joanne M. and Newman, Dianne K.},
abstractNote = {Arsenate respiration by bacteria was discovered over two decades ago and is catalyzed by diverse organisms using the well-conserved Arr enzyme complex. Until now, the mechanisms underpinning this metabolism have been relatively opaque. Here, we report the structure of an Arr complex (solved by X-ray crystallography to 1.6-Å resolution), which was enabled by an improved Arr expression method in the genetically tractable arsenate respirer Shewanella sp. ANA-3. We also obtained structures bound with the substrate arsenate (1.8 Å), the product arsenite (1.8 Å), and the natural inhibitor phosphate (1.7 Å). The structures reveal a conserved active-site motif that distinguishes Arr [(R/K)GRY] from the closely related arsenite respiratory oxidase (Arx) complex (XGRGWG). Arr activity assays using methyl viologen as the electron donor and arsenate as the electron acceptor display two-site ping-pong kinetics. A Mo(V) species was detected with EPR spectroscopy, which is typical for proteins with a pyranopterin guanine dinucleotide cofactor. Arr is an extraordinarily fast enzyme that approaches the diffusion limit ( K m = 44.6 ± 1.6 μM, k cat = 9,810 ± 220 seconds −1 ), and phosphate is a competitive inhibitor of arsenate reduction ( K i = 325 ± 12 μM). These observations, combined with knowledge of typical sedimentary arsenate and phosphate concentrations and known rates of arsenate desorption from minerals in the presence of phosphate, suggest that ( i ) arsenate desorption limits microbiologically induced arsenate reductive mobilization and ( ii ) phosphate enhances arsenic mobility by stimulating arsenate desorption rather than by inhibiting it at the enzymatic level.},
doi = {10.1073/pnas.1807984115},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 37,
volume = 115,
place = {United States},
year = {2018},
month = {8}
}

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DOI: 10.1073/pnas.1807984115

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