Identification of the molybdenum cofactor in chlorate-resistant mutants of Escherichia coli.
Experiments were performed to determine whether defects in molybdenum cofactor metabolism were responsible for the pleiotropic loss of the molybdoenzymes nitrate reductase and formate dehydrogenase in chl mutants of Escherichia coli. In wild-type E. coli, molybdenum cofactor activity was present in both the soluble and membrane-associated fractions when the cells were grown either aerobically or anaerobically, with and without nitrate. Molybdenum cofactor in the soluble fraction decreased when the membrane-bound nitrate reductase and formate dehydrogenase were induced. In the chl mutants, molybdenum cofactor activity was found in the soluble fraction of chlA, chlB, chlC, chlD, chlE, and chlG, but only chlB, chlC, chlD, and chlG expressed cofactor activity in the membrane fraction. The defect in the chlA mutants which prevented incorporation of the soluble cofactor into the membrane also caused the soluble cofactor to be defective in its ability to bind molybdenum. This cofactor was not active in the  absence of molybdate, and it required at least threefold more molybdate than did the wild type in the Neurospora crassa nit-1 complementation assay. However, the cofactor from the chlA strain mediated the dimerization of the nit-1 subunits in the presence and absence of molybdate to yield the 7.9S dimer. Growth of chlA mutants in medium with increased molybdate did not repair the defect in the chlA cofactor nor restore the molybdoenzyme activities. Thus, molybdenum cofactor was synthesized in all the chl mutants, but additional processing steps may be missing in chlA and chlE mutants for proper insertion of cofactor in the membrane.
D002704:Chlorates / Q000494:pharmacology*; D003067:Coenzymes; D004352:Drug Resistance, Microbial; D004790:Enzyme Induction; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005560:Formate Dehydrogenases / Q000096:biosynthesis; D008667:Metalloproteins; D008982:Molybdenum / Q000378:metabolism* / Q000494:pharmacology; D000090006:Molybdenum Cofactors; D009154:Mutation; D009565:Nitrate Reductases / Q000096:biosynthesis; D011621:Pteridines / Q000378:metabolism*
D002704:Chlorates; D003067:Coenzymes; D005560:Formate Dehydrogenases; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D009565:Nitrate Reductases; D011621:Pteridines; C022261:molybdopterin