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

Glasser, Nathaniel R. and Oyala, Paul H. and Osborne, Thomas H. and Santini, Joanne M. and Newman, Dianne K. (2018) Structural and mechanistic analysis of the arsenate respiratory reductase provides insight into environmental arsenic transformations. Proceedings of the National Academy of Sciences of the United States of America, 115 (37). E8614-E8623. ISSN 0027-8424. PMCID PMC6140538. doi:10.1073/pnas.1807984115.

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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.

Item Type:Article
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URLURL TypeDescription Information InCommentary CentralArticle
Glasser, Nathaniel R.0000-0002-2833-5166
Oyala, Paul H.0000-0002-8761-4667
Newman, Dianne K.0000-0003-1647-1918
Additional Information:© 2018 The Author(s). Published under the PNAS license. Edited by Joan Selverstone Valentine, University of California, Los Angeles, CA, and approved July 13, 2018 (received for review May 9, 2018) This research is rooted in work performed in the laboratory of Francois M. M. Morel (Massachusetts Institute of Technology/Princeton). We thank Shu-ou Shan and Lisa Racki for assistance with enzyme kinetics, Barbara Schoepp-Cothenet for correspondence about the phylogeny of Arr and Arx, and Jonas Peters for helpful discussions. The Caltech Molecular Observatory provided essential training, knowledge, and equipment for the crystallography in this work. NIH Grant 1R01AI127850-01A1 (to D.K.N.) supported this research. The Caltech EPR Facility was supported by National Science Foundation Grant 1531940 and the Dow Next Generation Educator Fund. This research used resources of the Advanced Light Source, which is a US Department of Energy (DOE) Office of Science User Facility under Contract DE-AC02-05CH11231. The Molecular Observatory is supported by the Gordon and Betty Moore Foundation, the Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US DOE Office of Science, Office of Basic Energy Sciences, under Contract DE-AC02-76SF00515. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the US DOE Office of Biological and Environmental Research, and by the NIH–National Institute of General Medical Sciences (including Grant P41GM103393). Funding to J.M.S. was provided by Biotechnology and Biological Sciences Research Council Grant BB/N012674/1. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of National Institute of General Medical Sciences or NIH. Author contributions: N.R.G., J.M.S., and D.K.N. designed research; N.R.G., P.H.O., T.H.O., and D.K.N. performed research; P.H.O., T.H.O., and J.M.S. contributed new reagents/analytic tools; N.R.G. and P.H.O. analyzed data; and N.R.G. and D.K.N. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, (PDB ID codes 6CZ7–6CZ9 and 6CZA). This article contains supporting information online at
Funding AgencyGrant Number
Dow Next Generation Educator FundUNSPECIFIED
Department of Energy (DOE)DE-AC02-05CH11231
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Caltech Sanofi-Aventis Bioengineering Research ProgramUNSPECIFIED
Department of Energy (DOE)DE-AC02-76SF00515
Biotechnology and Biological Sciences Research Council (BBSRC)BB/N012674/1
Subject Keywords:bacterial arsenate respiration; ArrAB; structure; enzymology; biogeochemistry
Issue or Number:37
PubMed Central ID:PMC6140538
Record Number:CaltechAUTHORS:20180813-133514351
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Official Citation:Structural and mechanistic analysis of the arsenate respiratory reductase provides insight into environmental arsenic transformations Nathaniel R. Glasser, Paul H. Oyala, Thomas H. Osborne, Joanne M. Santini, Dianne K. Newman Proceedings of the National Academy of Sciences Sep 2018, 115 (37) E8614-E8623; DOI: 10.1073/pnas.1807984115
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:88789
Deposited By: George Porter
Deposited On:13 Aug 2018 20:55
Last Modified:16 Nov 2021 00:29

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