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Properties of the periplasmic nitrate reductases from Paracoccus pantotrophus and Escherichia coli after growth in tungsten-supplemented media

Lookup NU author(s): Dr Clive Butler

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Abstract

Paracoccus pantotrophus grown anaerobically under denitrifying conditions expressed similar levels of the periplasmic nitrate reductase (NAP) when cultured in molybdate- or tungstate-containing media. A native PAGE gel stained for nitrate reductase activity revealed that only NapA from molybdate-grown cells displayed readily detectable nitrate reductase activity. Further kinetic analysis showed that the periplasmic fraction from cells grown on molybdate (3 μM) reduced nitrate at a rate of Vmax=3.41±0.16 μmol [NO3-] min-1 mg-1 with an affinity for nitrate of Km=0.24±0.05 mM and was heat-stable up to 50°C. In contrast, the periplasmic fraction obtained from cells cultured in media supplemented with tungstate (100 μM) reduced nitrate at a much slower rate, with much lower affinity (Vmax=0.05±0.002 μmol [NO3-] min-1 mg-1 and Km=3.91±0.45 mM) and was labile during prolonged incubation at >20°C. Nitrate-dependent growth of Escherichia coli strains expressing only nitrate reductase A was inhibited by sub-mM concentrations of tungstate in the medium. In contrast, a strain expressing only NAP was only partially inhibited by 10 mM tungstate. However, none of the above experimental approaches revealed evidence that tungsten could replace molybdenum at the active site of E. coli NapA. The combined data show that tungsten can function at the active site of some, but not all, molybdoenzymes from mesophilic bacteria. © 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Publication metadata

Author(s): Gates AJ, Hughes RO, Sharp SR, Millington PD, Nilavongse A, Cole JA, Leach E-R, Jepson B, Richardson DJ, Butler CS

Publication type: Article

Publication status: Published

Journal: FEMS Microbiology Letters

Year: 2003

Volume: 220

Issue: 2

Pages: 261-269

ISSN (print): 0378-1097

ISSN (electronic): 1574-6968

Publisher: Wiley-Blackwell

URL: http://dx.doi.org/10.1016/S0378-1097(03)00122-8

DOI: 10.1016/S0378-1097(03)00122-8

PubMed id: 12670690

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