title: The product of the molybdenum cofactor gene mobB of Escherichia coli is a GTP-binding protein.
abstract: The mob mutants of Escherichia coli are pleiotropically defective in molybdoenzyme activities because they are unable to catalyse the conversion of molybdopterin guanine dinucleotide, the active form of the molybdenum cofactor. The mob locus comprises two genes. The product of mobA, protein FA, has previously been purified to homogeneity and is able to restore molybdoenzyme activities following incubation with cell extracts of mob strains. The mobB gene, although not essential for the biosynthesis of active molybdoenzymes, encodes a protein which, sequence analysis strongly suggests, contains a nucleotide-binding site. We have overproduced the products of both the mobA and mobB genes in engineered E. coli strains and purified each to homogeneity. The preparation of protein FA (MobA) is simpler than that previously published and produces a much greater yield of active protein. The isolated MobB protein, which is dimeric in solution, acts in the presence of protein FA, to enhance the level of nitrate reductase activation achieved on incubation with mob cell extracts. Equilibrium dialysis experiments show that purified MobB binds 0.83 mol GTP/mol protein with a Kd of 2.0 microM. Isolated MobB also catalyses a low GTPase activity (turnover number of 3 x 10(-3) min-1) with a K(m) for GTP to GDP of 7.5 microM. Under the conditions tested, protein FA did not affect the GTP-binding or GTPase activity of MobB. Intrinsic (tryptophan) protein fluorescence measurements show that MobB also binds the nucleotides ATP, TTP and GDP, but with lower affinity than GTP. These results are consistent with a model whereby MobB binds the guanine nucleotide which is attached to molybdopterin during the biosynthesis of the molybdenum cofactor.
authors.affiliation: Department of Biochemistry, University of Dundee, UK.
authors.forename: D J
authors.initials: DJ
authors.lastname: Eaves
authors.forename: T
authors.initials: T
authors.lastname: Palmer
authors.forename: D H
authors.initials: DH
authors.lastname: Boxer
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry; D004789:Enzyme Activation; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C490938:MobB protein, E coli; C099206:MobB protein, bacteria; D015534:Trans-Activators; C095754:mobA protein, E coli; D006160:Guanosine Triphosphate; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins