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.

FIGURE 1.

Domain structure and nucleotide-binding motifs of MobA and MocA. A, surface structure MobA (Protein Data Bank code 1FRW) with the nucleotide binding domain (green) and the MPT binding domain (blue). Bound GTP is shown as sticks, and the M1 and M2 motifs are highlighted. B, GTP-binding site of MobA with the highlighted motifs M1 and M2 involved in coordination of the purine base. C, schematic overview of the two domains of the E. coli MobA and MocA with the two conserved motifs M1 and M2 involved in binding the base of the nucleotide. The generated MocA and MobA variants are listed in addition to their given names as cited in the text. The domains originating from MocA are highlighted in gray, and the domains originating from MobA are presented in white.

FIGURE 2.

Phylogenetic tree of MobA and MocA homologues. Protein phylogeny of MobA and MocA homologues is based on a full-length sequence alignment. The tree was constructed by the neighbor-joining method from a matrix of estimated numbers of amino acid substitutions per site calculated with the Dayhoff option of Phylip. The scale bar indicates 0.1 substitution per site. Names of organisms with only a single homologue are underlined; class, b, Bacilli; c, Clostridia; α, Alphaproteobacteria; β, Betaproteobacteria; γ, Gammaproteobacteria; δ, Deltaproteobacteria.

FIGURE 3.

Purification of MocA and MobA variants after expression in E. coli BL21(DE3) cells. 12% SDS-PAGE analysis of purified MocA and MobA variants is shown. 10 μg of MocA and MobA variants after Ni-NTA or Ni-TED affinity chromatography, respectively, is shown. Lane I, MocA; lane II, MocA-M1; lane III, MocA-M2; lane IV, MocA-M1-M2; lane V, N-MobA-C-MocA; lane VI, molecular weight marker; lane VII, N-MocA-C-MobA; lane VIII, MobA-M1-M2; lane IX, MobA-M1; lane X, MobA-M2, lane XI, MobA.

FIGURE 4.

MobA-GTP complex and modeled MocA-CTP complex. A, key interactions between MobA and GTP are shown (Protein Data Bank code 1 FRW). GTP is shown in green. Residues involved in nucleotide and GTP binding are shown in all-bonds representation. B, model of the key interactions between MocA and CTP (structure was modeled using the coordinates from MobA using the program COOT). CTP is shown in orange. Residues involved in nucleotide and CTP binding are shown in all-bonds representation.