Molybdenum cofactor requirement for in vitro activation of apo-molybdoenzymes of Escherichia coli.
The apo-nitrate reductase precursor in an Escherichia coli chlB mutant preparation obtained following growth in the presence of tungstate is activated by incubation with protein FA and a heat-treated preparation from an E. coli crude extract. We show that the requirement for heat-treated E. coli crude extract can be fulfilled by material obtained from either of two heat-denatured purified E. coli molybdoenzymes, namely nitrate reductase or trimethylamine N-oxide reductase. Apo-trimethylamine N-oxide reductase precursor in the tungstate-grown chlB preparation can be activated in a similar manner with material from either heat-denatured molybdoenzyme. The active component in the denatured molybdoenzyme preparations is shown to be the molybdenum cofactor by Neurospora crassa nit1 molybdenum cofactor assay, size estimation and fluorimetric analysis. The direct demonstration of the requirement for molybdenum cofactor in the E. coli tungstate-grown chlB complementation system is an important step towards the molecular definition of the activation process and an understanding of the mechanism of cofactor acquisition during molybdoenzyme biosynthesis.
Giordano
G
G
Départment de Biologie, Faculté de Sciences de Luminy, Marseille, France.
Boxer
D H
DH
Pommier
J
J
D003067:Coenzymes; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D005816:Genetic Complementation Test; D006358:Hot Temperature; D008667:Metalloproteins / Q000378:metabolism*; D000090006:Molybdenum Cofactors; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009492:Neurospora crassa / Q000378:metabolism; D009565:Nitrate Reductases / Q000378:metabolism*; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011621:Pteridines / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014414:Tungsten / Q000494:pharmacology; D017973:Tungsten Compounds
D003067:Coenzymes; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D009247:NADH, NADPH Oxidoreductases; D009565:Nitrate Reductases; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011621:Pteridines; D014414:Tungsten; D017973:Tungsten Compounds; C022261:molybdopterin; C021567:methylamine dehydrogenase; C045951:tungstate