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Applied and Environmental Microbiology, August 2006, p. 5173-5180, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00568-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Resolution of Distinct Membrane-Bound Enzymes from Enterobacter cloacae SLD1a-1 That Are Responsible for Selective Reduction of Nitrate and Selenate Oxyanions

Institute for Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne NE2 4HH, United Kingdom,¹ School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom²

Received 9 March 2006/ Accepted 15 May 2006

Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with and ß subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (ß) complex with an apparent molecular mass of 600 kDa. The individual subunit sizes are 100 kDa (), 55 kDa (ß), and 36 kDa (), with a predicted overall subunit composition of ₃ß₃₃. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent K_m for selenate was determined to be 2 mM, with an observed V_max of 500 nmol SeO₄^2– min^–1 mg^–1 (k_cat, 5.0 s^–1). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.

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