Isolation of protein FA, a product of the mob locus required for molybdenum cofactor biosynthesis in Escherichia coli.
The mob mutants in Escherichia coli are pleiotropically defective in all molybdoenzyme activities. They synthesise molybdopterin, the unique core of the molybdenum cofactor, but are unable to attach the GMP moiety to molybdopterin to form molybdopterin guanine dinucleotide, the functional molybdenum cofactor in Escherichia coli. A partially purified preparation termed protein FA (protein factor d'association), is able to restore molybdoenzyme activities to broken cell preparations of mob mutants. A fragment of DNA capable of complementing mob mutants has been isolated from an E. coli genomic library. Strains carrying this DNA in a multicopy plasmid, express 30-fold more protein FA activity than the wild-type bacterium. Protein FA has been purified to homogeneity by a combination of ion-exchange, affinity and gel-filtration chromatography. Protein FA consists of a single polypeptide of molecular mass 22 kDa and is monomeric in solution. N-terminal amino acid sequencing confirmed that protein FA is a product of the first gene at the mob locus. The purified protein FA was required in stoichiometric rather than catalytic amounts in the process that leads to the activation of the precursor of the molybdoenzyme nitrate reductase, which is consistent with the requirement of a further component in the activation.
Department of Biochemistry, Medical Sciences Institute, Dundee University, Scotland.
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T
Palmer
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Vasishta
P W
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Whitty
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Boxer
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003067:Coenzymes; D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005798:Genes, Bacterial; D015698:Genomic Library; D005838:Genotype; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000090006:Molybdenum Cofactors; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism; D010957:Plasmids; D011621:Pteridines / Q000378:metabolism*
D001426:Bacterial Proteins; D003067:Coenzymes; D004269:DNA, Bacterial; D004792:Enzyme Precursors; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D050901:Nitrate Reductase; D009565:Nitrate Reductases; D011621:Pteridines; C022261:molybdopterin