Cited by 53Metalloenzymes and iron-sulfur proteinsBacterial Genetics and BiotechnologyWastewater Treatment and Nitrogen Removal

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

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Gates, Andrew J, Hughes, Richard O, Sharp, Sarah R, Millington, Paul D, Nilavongse, Arjaree, Cole, Jeffrey A, Leach, Emily-Rose, Jepson, Brian +2 more·2003·FEMS Microbiology Letters

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 microM) reduced nitrate at a rate of V(max)=3.41+/-0.16 micromol [NO(3)(-)] min(-1) mg(-1) with an affinity for nitrate of K(m)=0.24+/-0.05 mM and was heat-stable up to 50 degrees C. In contrast, the periplasmic fraction obtained from cells cultured in media supplemented with tungstate (100 microM) reduced nitrate at a much slower rate, with much lower affinity (V(max)=0.05+/-0.002 micromol [NO(3)(-)] min(-1) mg(-1) and K(m)=3.91+/-0.45 mM) and was labile during prolonged incubation at >20 degrees 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.

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  1. Nicholls, D.G. and Ferguson, S.J. (2002) Bioenergetics 3. Elsevier Science, London.

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Authors
Gates, A. J., Hughes, R. O., Sharp, S. R., Millington, P. D., Nilavongse, A., Cole, J. A., Leach, E.-R., Jepson, B., Richardson, D. J., & Butler, C. S.
Affiliations
  • Newcastle University (GB)
  • University of Birmingham (GB)
  • University of East Anglia (GB)
Corresponding author
Butler, Clive S
Venue
FEMS Microbiology Letters
Published
2003-03
Volume
220
Issue
2
Pages
261-269
ISSN
  • 0378-1097 (ISSN-L)
  • 1574-6968
OpenAlex
W2022642927
Cited by
53
References
39
Citations by year
2026: 3 · 2025: 1 · 2024: 2 · 2023: 4 · 2022: 1 · 2021: 4 · 2020: 2 · 2019: 4 · 2018: 2 · 2017: 1 · 2015: 3 · 2014: 1 · 2013: 1 · 2012: 4
FWCI
2.28
Type
journal article
Language
en
Open access
Locations
  • FEMS Microbiology Letters (publishedVersion)
  • PubMed (publishedVersion)
  • University of Birmingham Research Portal (University of Birmingham) (submittedVersion)
  • UEA Digital Repository (University of East Anglia) (submittedVersion)
  • Newcastle University ePrints (Newcastle Univesity)
Funders
  • University of Oxford
  • Biotechnology and Biological Sciences Research Council
Topics
  • Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Metalloenzymes and iron-sulfur proteins
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Physical Sciences › Environmental Science › Pollution › Wastewater Treatment and Nitrogen Removal
Keywords
periplasmic nitrate reductase, napA, tungstate, molybdenum, tungsten substitution, molybdoenzymes, Paracoccus pantotrophus, Escherichia coli, denitrification, enzyme kinetics, enzyme stability, tungstate inhibition, anaerobic bacteria
MeSH terms
Anaerobiosis, Culture Media, Escherichia coli, Molybdenum, Nitrate Reductases, Nitrates, Oxidation-Reduction, Paracoccus, Tungsten, Gene Expression Regulation, Bacterial, Tungsten Compounds

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