Molybdenum incorporation in tungsten aldehyde oxidoreductase enzymes from Pyrococcus furiosus.
The hyperthermophilic archaeon Pyrococcus furiosus expresses five aldehyde oxidoreductase (AOR) enzymes, all containing a tungsto-bispterin cofactor. The growth of this organism is fully dependent on the presence of tungsten in the growth medium. Previous studies have suggested that molybdenum is not incorporated in the active site of these enzymes. Application of the radioisotope (99)Mo in metal isotope native radioautography in gel electrophoresis (MIRAGE) technology to P. furiosus shows that molybdenum can in fact be incorporated in all five AOR enzymes. Mo(V) signals characteristic for molybdopterin were observed in formaldehyde oxidoreductase (FOR) in electron paramagnetic resonance (EPR)-monitored redox titrations. Our finding that the aldehyde oxidation activity of FOR and WOR5 (W-containing oxidoreductase 5) correlates only with the residual tungsten content suggests that the Mo-containing AORs are most likely inactive. An observed W/Mo antagonism is indicative of tungstate-dependent negative feedback of the expression of the tungstate/molybdate ABC transporter. An intracellular selection mechanism for tungstate and molybdate processing has to be present, since tungsten was found to be preferentially incorporated into the AORs even under conditions with comparable intracellular concentrations of tungstate and molybdate. Under the employed growth conditions of starch as the main carbon source in a rich medium, no tungsten- and/or molybdenum-associated proteins are detected in P. furiosus other than the high-affinity transporter, the proteins of the metallopterin insertion machinery, and the five W-AORs.
Sevcenco
Ana-Maria
AM
Department of Biotechnology, Delft University of Technology, Delft, Netherlands.
Bevers
Loes E
LE
Pinkse
Martijn W H
MW
Krijger
Gerard C
GC
Wolterbeek
Hubert T
HT
Verhaert
Peter D E M
PD
Hagen
Wilfred R
WR
Hagedoorn
Peter-Leon
PL
D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D019843:Archaeal Proteins / Q000378:metabolism*; D001345:Autoradiography; D003067:Coenzymes / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D008982:Molybdenum / Q000378:metabolism*; D010084:Oxidation-Reduction; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000378:metabolism; D011868:Radioisotopes / Q000378:metabolism; D013194:Staining and Labeling; D014414:Tungsten / Q000378:metabolism*
D019843:Archaeal Proteins; D003067:Coenzymes; D011868:Radioisotopes; D008982:Molybdenum; D000445:Aldehyde Oxidoreductases; D014414:Tungsten