Publications

• The crystal structure of the Escherichia coli MobA protein provides insight into molybdopterin guanine dinucleotide biosynthesis.

  Lake M.W., Temple C.A., Rajagopalan K.V., Schindelin H.

  The molybdenum cofactor (Moco) is found in a variety of enzymes present in all phyla and comprises a family of related molecules containing molybdopterin (MPT), a tricyclic pyranopterin with a cis-dithiolene group, as the invariant essential moiety. MPT biosynthesis involves a conserved pathway, b ... >> More

  The molybdenum cofactor (Moco) is found in a variety of enzymes present in all phyla and comprises a family of related molecules containing molybdopterin (MPT), a tricyclic pyranopterin with a cis-dithiolene group, as the invariant essential moiety. MPT biosynthesis involves a conserved pathway, but some organisms perform additional reactions that modify MPT. In eubacteria, the cofactor is often present in a dinucleotide form combining MPT and a purine or pyrimidine nucleotide via a pyrophosphate linkage. In Escherichia coli, the MobA protein links a guanosine 5'-phosphate to MPT forming molybdopterin guanine dinucleotide. This reaction requires GTP, MgCl(2), and the MPT form of the cofactor and can efficiently reconstitute Rhodobacter sphaeroides apo-DMSOR, an enzyme that requires molybdopterin guanine dinucleotide for activity. In this paper, we present the crystal structure of MobA, a protein containing 194 amino acids. The MobA monomer has an alpha/beta architecture in which the N-terminal half of the molecule adopts a Rossman fold. The structure of MobA has striking similarity to Bacillus subtilis SpsA, a nucleotide-diphospho-sugar transferase involved in sporulation. The cocrystal structure of MobA and GTP reveals that the GTP-binding site is located in the N-terminal half of the molecule. Conserved residues located primarily in three signature sequence motifs form crucial interactions with the bound nucleotide. The binding site for MPT is located adjacent to the GTP-binding site in the C-terminal half of the molecule, which contains another set of conserved residues presumably involved in MPT binding. << Less

  J. Biol. Chem. 275:40211-40217(2000) [PubMed] [EuropePMC]

• Biochemical and structural analysis of the molybdenum cofactor biosynthesis protein MobA.

  Guse A., Stevenson C.E., Kuper J., Buchanan G., Schwarz G., Giordano G., Magalon A., Mendel R.R., Lawson D.M., Palmer T.

  Molybdopterin guanine dinucleotide (MGD) is the form of the molybdenum cofactor that is required for the activity of most bacterial molybdoenzymes. MGD is synthesized from molybdopterin (MPT) and GTP in a reaction catalyzed by the MobA protein. Here we report that wild type MobA can be copurified ... >> More

  Molybdopterin guanine dinucleotide (MGD) is the form of the molybdenum cofactor that is required for the activity of most bacterial molybdoenzymes. MGD is synthesized from molybdopterin (MPT) and GTP in a reaction catalyzed by the MobA protein. Here we report that wild type MobA can be copurified along with bound MPT and MGD, demonstrating a tight binding of both its substrate and product. To study structure-function relationships, we have constructed a number of site-specific mutations of the most highly conserved amino acid residues of the MobA protein family. Variant MobA proteins were characterized for their ability to support the synthesis of active molybdenum enzymes, to bind MPT and MGD, to interact with the molybdenum cofactor biosynthesis proteins MobB and MoeA. They were also characterized by x-ray structural analysis. Our results suggest an essential role for glycine 15 of MobA, either for GTP binding and/or catalysis, and an involvement of glycine 82 in the stabilization of the product-bound form of the enzyme. Surprisingly, the individual and double substitution of asparagines 180 and 182 to aspartate did not affect MPT binding, catalysis, and product stabilization. << Less

  J. Biol. Chem. 278:25302-25307(2003) [PubMed] [EuropePMC]

• Mechanism of assembly of the bis(molybdopterin guanine dinucleotide)molybdenum cofactor in Rhodobacter sphaeroides dimethyl sulfoxide reductase.

  Temple C.A., Rajagopalan K.V.

  A fully defined in vitro system has been developed for studying the mechanism of assembly of the bis(molybdopterin guanine dinucleotide)molybdenum cofactor in Rhodobacter sphaeroides dimethyl sulfoxide reductase (DMSOR). R. sphaeroides DMSOR expressed in a mobA(-) Escherichia coli strain lacks mol ... >> More

  A fully defined in vitro system has been developed for studying the mechanism of assembly of the bis(molybdopterin guanine dinucleotide)molybdenum cofactor in Rhodobacter sphaeroides dimethyl sulfoxide reductase (DMSOR). R. sphaeroides DMSOR expressed in a mobA(-) Escherichia coli strain lacks molybdopterin and molybdenum but contains a full complement of guanine in the form of GMP and GDP. Escherichia coli MobA, molybdopterin-Mo, GTP, and MgCl(2) are required and sufficient for the in vitro activation of purified DMSOR expressed in the absence of MobA. High levels of MobA inhibit the in vitro activation. A chaperone is not required for the in vitro activation process. The reconstituted DMSOR can exhibit up to 73% of the activity observed in recombinant DMSOR purified from a wild-type strain. The use of radiolabeled GTP has demonstrated incorporation of the guanine moiety from the GTP into the activated DMSOR. No role was observed for E. coli MobB in the in vitro activation of apo-DMSOR. This work also represents the first time that the MobA-mediated conversion of molybdopterin to molybdopterin guanine dinucleotide has been demonstrated directly without using the activation of a molybdoenzyme as an indicator for cofactor formation. << Less

  J. Biol. Chem. 275:40202-40210(2000) [PubMed] [EuropePMC]