318 Metallomics, 2013, 5, 318--324 This journal is c The Royal Society of Chemistry 2013 Cite this: Metallomics, 2013, 5, 318 Cold-adapted arsenite oxidase from a psychrotolerant Polaromonas species† Thomas H. Osborne, a Matthew D. Heath, a Andrew C. R. Martin, a Jaroslaw A. Pankowski, a Karen A. Hudson-Edwards b and Joanne M. Santini* a Polaromonas sp. str. GM1 is an aerobic, psychrotolerant, heterotrophic member of the Betaproteobacteria and is the only isolate capable of oxidising arsenite at temperatures below 10 1C. Sequencing of the aio gene cluster in GM1 revealed the presence of the aioB and aioA genes, which encode the arsenite oxidase but the regulatory genes typically found upstream of aioB in other members of the Proteobacteria were absent. The GM1 Aio was purified to homogeneity and was found to be a heterodimer. The enzyme contained Mo and Fe as cofactors and had, using the artificial electron acceptor 2,6-dichloro- phenolindophenol, a K m for arsenite of 111.70 Æ 0.88 mM and a V max of 12.16 Æ 0.30 U mg À1 , which is the highest reported specific activity for any known Aio. The temperature-activity profiles of the arsenite oxidases from GM1 and the mesophilic betaproteobacterium Alcaligenes faecalis were compared and showed that the GM1 Aio was more active at low temperatures than that of A. faecalis. A homology model of the GM1 Aio was made using the X-ray crystal structure of the Aio from A. faecalis as the template. Structural changes that account for cold adaptation were identified and it was found that these resulted in increased enzyme flexibility and a reduction in the hydrophobicity of the core. Introduction Arsenic is a group 5 metalloid and is renowned for its toxicity. 1 The soluble inorganic forms of arsenic, arsenite (+3) and arsenate (+5), are widespread in terrestrial and aquatic environments, 2 where arsenite is the more toxic and mobile species. 3 The consumption of naturally occurring inorganic arsenic in drinking water has caused poisoning of human populations across regions of West Bengal, Bangladesh, Chile, Taiwan and Vietnam, while anthro- pogenic activity (principally through the mining of valuable metals) has resulted in arsenic contamination of environments in Canada, USA, Argentina and Australia. 4,5 Despite its toxicity, certain microbes are capable of gaining energy by oxidising arsenite to arsenate, 6 and this process contributes to the global biogeochemical cycling of arsenic. 7 Since the first discovery of bacterial arsenite oxidation by Green in 1918, 8 over 50 strains of arsenite-oxidising bacteria have been isolated. The arsenite-oxidising bacteria are phylogenetically diverse and have been isolated from different environments (reviewed by Osborne and Santini). 9 Aerobic oxidation of arsenite is catalysed by arsenite oxidase (Aio) (see Lett et al. for recent changes to the nomenclature for the arsenite oxidase). 10 Aio is a member of the DMSO reductase family of molybdoenzymes which is a family of redox enzymes that function in a diverse range of prokaryotic electron transport chains. 11 The enzyme is a heterodimer that consists of a large a-subunit, AioA and a small b-subunit, AioB. The X-ray crystal structure of the Aio from the mesophilic, heterotrophic betaproteo- bacterium Alcaligenes faecalis has been resolved. 12 The structure shows that AioA contains a 3Fe–4S cluster and a bis-molybdopterin guanine dinucleotide ( bis-MGD) cofactor at the active site, while AioB contains a 2Fe–2S Rieske cluster. The Aio is exported to the periplasm by the Twin-arginine translocation (Tat) pathway conferred by an N-terminal signal sequence on AioB. The Aio that have been characterised to date have all been purified from mesophilic arsenite oxidisers (reviewed by Heath et al. ). 13 The aio operon structure always includes the aioA and aioB genes, with aioB located upstream of aioA. In some arsenite oxidisers of the Alpha- and Betaproteobacteria a cytochrome c-encoding gene (cytC) is located downstream of aioA 9 and this cytochrome has been shown to serve as the electron acceptor to the Aio. 14,15 Often found upstream of aioB are three genes (aioXSR) whose products are involved in the regulation of arsenite oxidase gene expression. AioS is a sensor histidine kinase and AioR a response regulator. 16 Together with a third a Institute of Structural and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK. E-mail: j.santini@ucl.ac.uk b Department of Earth and Planetary Sciences, Birkbeck, University of London, Malet Street, London, WC1E 7HX, UK † Electronic supplementary information (ESI) available. See DOI: 10.1039/c2mt20180a Received 7th September 2012, Accepted 30th October 2012 DOI: 10.1039/c2mt20180a www.rsc.org/metallomics Metallomics PAPER Open Access Article. Published on 31 October 2012. Downloaded on 10/8/2021 8:39:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online View Journal | View Issue