title: Molybdopterin dinucleotide biosynthesis in Escherichia coli: identification of amino acid residues of molybdopterin dinucleotide transferases that determine specificity for binding of guanine or cytosine nucleotides.
abstract: The molybdenum cofactor is modified by the addition of GMP or CMP to the C4' phosphate of molybdopterin forming the molybdopterin guanine dinucleotide or molybdopterin cytosine dinucleotide cofactor, respectively. The two reactions are catalyzed by specific enzymes as follows: the GTP:molybdopterin guanylyltransferase MobA and the CTP:molybdopterin cytidylyltransferase MocA. Both enzymes show 22% amino acid sequence identity and are specific for their respective nucleotides. Crystal structure analysis of MobA revealed two conserved motifs in the N-terminal domain of the protein involved in binding of the guanine base. Based on these motifs, we performed site-directed mutagenesis studies to exchange the amino acids to the sequence found in the paralogue MocA. Using a fully defined in vitro system, we showed that the exchange of five amino acids was enough to obtain activity with both GTP and CTP in either MocA or MobA. Exchange of the complete N-terminal domain of each protein resulted in the total inversion of nucleotide specificity activity, showing that the N-terminal domain determines nucleotide recognition and binding. Analysis of protein-protein interactions showed that the C-terminal domain of either MocA or MobA determines the specific binding to the respective acceptor protein.
authors.affiliation: Department of Molecular Enzymology, Institute of Biochemistry and Biology, University of Potsdam, 14476 Potsdam, Germany.
authors.forename: Meina
authors.initials: M
authors.lastname: Neumann
authors.forename: Farida
authors.initials: F
authors.lastname: Seduk
authors.forename: Chantal
authors.initials: C
authors.lastname: Iobbi-Nivol
authors.forename: Silke
authors.initials: S
authors.lastname: Leimkühler
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000378:metabolism; D003596:Cytosine / Q000378:metabolism; D003597:Cytosine Nucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006147:Guanine / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011622:Pterins / Q000378:metabolism*; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
chemical_list: D003597:Cytosine Nucleotides; D029968:Escherichia coli Proteins; D006150:Guanine Nucleotides; D011622:Pterins; C095754:mobA protein, E coli; C066895:molybdopterin cytosine dinucleotide; C063761:molybdopterin guanine dinucleotide; D006147:Guanine; D003596:Cytosine; D000445:Aldehyde Oxidoreductases; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase; C555940:CTP-molybpterin cytiylyltransferase, E coli; D009713:Nucleotidyltransferases