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Health, Safety & Environment

Arsenite oxidase in complex with antimonite and arsenite oxyanions - insights into the catalytic mechanism

Arsenic contamination of groundwater is among one of the biggest health threats affecting millions of people in the world. There is an urgent need for efficient arsenic biosensors where the use of arsenic metabolizing enzymes can be explored. In this work we have solved the crystal structures of four complexes of arsenite oxidase Aio obtained in complex with arsenic and antimony oxyanions and the structures determined correspond to intermediate states of the enzymatic mechanism. These structural data were complemented with DFT calculations providing a unique view of the molybdenum  active site at different time points that, together with mutagenesis data, enabled to clarify the enzymatic mechanism and the molecular determinants for the oxidation of AsIII to the less toxic AsV species.

Engrola, M. A. S. Correia, C. Watson, C. C. Romão, L. F. Veiros, M. J. Romão, T. Santos-Silva, and J. M. Santini,Arsenite oxidase in complex with antimonite and arsenite oxyanions - insights into the catalytic mechanism, J Biol Chem, 2023, 105036.

In silico analysis of phylogeny, structure, and function of arsenite oxidase from unculturable microbiome of arsenic contaminated soil

Background Arsenite oxidase (EC is a metalloenzyme that catalyzes the oxidation of arsenite into lesser toxic arsenate. In this study, 78 amino acid sequences of arsenite oxidase from unculturable bacteria available in metagenomic data of arsenic-contaminated soil have been characterized by using standard bioinformatics tools to investigate its phylogenetic relationships, three-dimensional structure and functional parameters. Results The phylogenetic relationship of all arsenite oxidase from unculturable microorganisms was revealed their closeness to bacterial order Rhizobiales. The higher aliphatic content showed that these enzymes are thermostable and could be used for in situ bioremediation. A representative protein from each phylogenetic cluster was analysed for secondary structure arrangements which indicated the presence of alpha-helices (similar to 63%), beta-sheets (57-60%) and turns (13-15%). The validated 3D models suggested that these proteins are hetero-dimeric with two chains whereas alpha chain is the main catalytic subunit which binds with arsenic oxides. Three representative protein models were deposited in Protein Model Database. The query enzymes were predicted with two conserved motifs, one is Rieske 3Fe-4S and the other is molybdopterin protein. Conclusions Computational analysis of protein interactome revealed the protein partners might be involved in the whole process of arsenic detoxification by Rhizobiales. The overall report is unique to the best of our knowledge, and the importance of this study is to understand the theoretical aspects of the structure and functions of arsenite oxidase in unculturable bacteria residing in arsenic-contaminated sites.

S. Pal, and K. Sengupta,In silico analysis of phylogeny, structure, and function of arsenite oxidase from unculturable microbiome of arsenic contaminated soil, Journal of Genetic Engineering and Biotechnology, 2021, 19.

Arsenite Oxidase

Electron transfer through arsenite oxidase: Insights into Rieske interaction with cytochrome c

Arsenic is a widely distributed environmental toxin whose presence in drinking water poses a threat to > 140 million people worldwide. The respiratory enzyme arsenite oxidase from various bacteria catalyses the oxidation of arsenite to arsenate and is being developed as a biosensor for arsenite. The arsenite oxidase from Rhizobium sp. str. NT-26 (a member of the Alphaproteobacteria) is a heterotetramer consisting of a large catalytic subunit (AioA), which contains a molybdenum centre and a 3Fe-4S cluster, and a small subunit (AioB) containing a Rieske 2Fe-2S cluster. Stopped-flow spectroscopy and isothermal titration calorimetry (ITC) have been used to better understand electron transfer through the redox-active centres of the enzyme, which is essential for biosensor development. Results show that oxidation of arsenite at the active site is extremely fast with a rate of > 4000 s-1 and reduction of the electron acceptor is rate-limiting. An AioB-F108A mutation results in increased activity with the artificial electron acceptor DCPIP and decreased activity with cytochrome c, which in the latter as demonstrated by ITC is not due to an effect on the protein-protein interaction but instead to an effect on electron transfer. These results provide further support that the AioB F108 is important in electron transfer between the Rieske subunit and cytochrome c and its absence in the arsenite oxidases from the Betaproteobacteria may explain the inability of these enzymes to use this electron acceptor.

Watson, C., Niks, D., Hille, R., Vieira, M., Schoepp-Cothenet, B., Marques, A. T., Romao, M. J., Santos-Silva, T., and Santini, J. M.,Electron transfer through arsenite oxidase: Insights into Rieske interaction with cytochrome c, Biochimica Et Biophysica Acta-Bioenergetics, 2017, 1858, 865-872.

Arsenite oxidase from Alcaligenes faecalis NCIB 8687 is a molybdenum/iron protein involved in the detoxification of arsenic. It oxidizes arsenite [(AsO 2-)-O-III], which binds to essential sulfhydryl groups of proteins and dithiols, to the relatively less toxic arsenate [(AsO4 3-)-O-V]. Arsenite oxidase consists of a large subunit of 825 residues and a small subunit of approximately 134 residues. The large subunit contains a Mo site, consisting of a Mo atom bound to two pterin cofactors, and a [3Fe-4S] cluster. The large subunit of arsenite oxidase is similar to other members of the dimethylsulfoxide (DMSO) reductase family of molybdenum enzymes, particularly the dissimilatory periplasmic nitrate reductase from Desulfovibrio desulfuricans , but is unique in having no covalent bond between the polypeptide and the Mo atom. The small subunit has no counterpart among known Mo protein structures but is homologous to the Rieske [2Fe-2S] protein domain of the cytochrome be, and cytochrome b(6)f complexes and to the Rieske domain of naphthalene 1,2-dioxygenase

Ellis, P.J., Conrads, T., Hille, R., and Kuhn, P., Crystal structure of the 100 kDa arsenite oxidase from Alcaligenes faecalis in two crystal forms at 1.64 angstrom and 2.03 angstrom, Structure, 2001, 9, 125-132.

Lebrun, E., Brugna, M., Baymann, F., Muller, D., Lievremont, D., Lett, M. C., and Nitschke, W., Arsenite oxidase, an ancient bioenergetic enzyme, Molecular Biology and Evolution, 2003, 20, 686-693.

Users of the Database should be aware that inclusion of an abstract in the Database does not imply any IMOA endorsement of the accuracy or reliability of the reported data or the quality of a publication.