Properties of the periplasmic nitrate reductases from Paracoccus pantotrophus and Escherichia coli after growth in tungsten-supplemented media.
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.
Gates
Andrew J
AJ
School of Cell and Molecular Biosciences, The Medical School, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK.
Hughes
Richard O
RO
Sharp
Sarah R
SR
Millington
Paul D
PD
Nilavongse
Arjaree
A
Cole
Jeffrey A
JA
Leach
Emily-Rose
ER
Jepson
Brian
B
Richardson
David J
DJ
Butler
Clive S
CS
D000693:Anaerobiosis; D003470:Culture Media; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D008982:Molybdenum / Q000494:pharmacology; D009565:Nitrate Reductases / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D010084:Oxidation-Reduction; D010230:Paracoccus / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D014414:Tungsten / Q000494:pharmacology*; D017973:Tungsten Compounds / Q000494:pharmacology
D003470:Culture Media; D009566:Nitrates; D017973:Tungsten Compounds; C044659:molybdate; D008982:Molybdenum; D009565:Nitrate Reductases; C045951:tungstate; D014414:Tungsten