The History of the Discovery of the Molybdenum Cofactor and Novel Aspects of its Biosynthesis in Bacteria.

Biosynthesis of the molybdenum cofactor in bacteria is described with a detailed analysis of each individual reaction leading to the formation of stable intermediates during the synthesis of molybdopterin from GTP. As a starting point, the discovery of molybdopterin and the elucidation of its structure through the study of stable degradation products are described. Subsequent to molybdopterin synthesis, the molybdenum atom is added to the molybdopterin dithiolene group to form the molybdenum cofactor. This cofactor is either inserted directly into specific molybdoenzymes or is further modified by the addition of nucleotides to the molybdopterin phosphate group or the replacement of ligands at the molybdenum center.

Crystal structure of a molybdopterin synthase-precursor Z complex: insight into its sulfur transfer mechanism and its role in molybdenum cofactor deficiency.

In almost all biological life forms, molybdenum and tungsten are coordinated by molybdopterin (MPT), a tricyclic pyranopterin containing a cis-dithiolene group. Together, the metal and the pterin moiety form the redox reactive molybdenum cofactor (Moco). Mutations in patients with deficiencies in Moco biosynthesis usually occur in the enzymes catalyzing the first and second steps of biosynthesis, leading to the formation of precursor Z and MPT, respectively. The second step is catalyzed by the heterotetrameric MPT synthase protein consisting of two large (MoaE) and two small (MoaD) subunits with the MoaD subunits located at opposite ends of a central MoaE dimer. Previous studies have determined that the conversion of the sulfur- and metal-free precursor Z to MPT by MPT synthase involves the transfer of sulfur atoms from a C-terminal MoaD thiocarboxylate to the C-1' and C-2' positions of precursor Z. Here, we present the crystal structures of non-thiocarboxylated MPT synthase from Staphylococcus aureus in its apo form and in complex with precursor Z. A comparison of the two structures reveals conformational changes in a loop that participates in interactions with precursor Z. In the complex, precursor Z is bound by strictly conserved residues in a pocket at the MoaE dimer interface in close proximity of the C-terminal glycine of MoaD. Biochemical evidence indicates that the first dithiolene sulfur is added at the C-2' position.

Role of the C-terminal Gly-Gly motif of Escherichia coli MoaD, a molybdenum cofactor biosynthesis protein with a ubiquitin fold.

In Escherichia coli, the MoaD protein plays a central role in the conversion of precursor Z to molybdopterin (MPT) during molybdenum cofactor biosynthesis. MoaD has a fold similar to that of ubiquitin and contains a highly conserved C-terminal Gly-Gly motif, which in its active form contains a transferrable sulfur in the form of a thiocarboxylate group. During MPT biosynthesis, MoaD cycles between two different heterotetrameric complexes, one with MoaE to form MPT synthase and the other with MoeB, a protein similar to E1 in the ubiquitin pathway, to regenerate its transferrable sulfur. To determine the specific roles of each of the two terminal Gly residues with regard to the MoaD cycle, variants at the penultimate (Gly80) or terminal (Gly81) residues of both MoaD and thiocarboxylated MoaD were created. These variants were analyzed to determine their effects on complex formation with MoaE and MoeB, formation of the MoaD-acyl-adenylate complex, transfer of sulfur to precursor Z to form MPT, and total cofactor biosynthesis. The combined results show that while conservative substitutions at Gly80 had little effect on any of the processes that were examined, the terminal MoaD residue (Gly81) is important for transfer of sulfur to precursor Z and essential for formation of the MoaD-AMP complex. These results further our understanding of the mechanistic similarities of the MoaD-MoeB reaction to that of the ubiquitin-E1 system.

Thermodynamic analysis of subunit interactions in Escherichia coli molybdopterin synthase.

The molybdopterin (MPT) synthase complex in Escherichia coli consists of two MoaE subunits and two MoaD subunits in a heterotetrameric structure with the two MoaE subunits forming a central dimer. Each MoaD subunit binds to a single MoaE molecule to form two identical MoaE/MoaD interfaces. Here we define the thermodynamic properties of the interaction between MoaE and MoaD in MPT synthase using a H/D exchange and matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy based method termed SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX-derived protein folding free energies and m values are reported for MoaE in the presence and absence of MoaD and MoaD-SH, the thiocarboxylated form of MoaD that is essential for the catalytic activity of MPT synthase. The protein folding free energy measurements were used to calculate a dissociation constant of 17 +/- 7 microM for the binding of MoaD to MoaE in inactive MPT synthase and a dissociation constant of 2.6 +/- 0.9 microM for the binding of MoaD-SH to MoaE in active MPT synthase. The increased binding affinity of MoaD-SH for MoaE is consistent with a previously proposed mechanism for the MPT synthase reaction. Using the increased m values exhibited by MoaE in the presence of either MoaD subunit, the solvent accessible surface area buried upon formation of the subunit interface in MPT synthase was estimated to be 2378 A(2) for inactive MPT synthase and 4117 A(2) for active MPT synthase.

Mechanistic and mutational studies of Escherichia coli molybdopterin synthase clarify the final step of molybdopterin biosynthesis.

Biosynthesis of the molybdenum cofactor, a chelate of molybdenum or tungsten with a novel pterin, occurs in virtually all organisms including humans. In the cofactor, the metal is complexed to the unique cis-dithiolene moiety located on the pyran ring of molybdopterin. Escherichia coli molybdopterin synthase, the protein responsible for adding the dithiolene to a desulfo precursor termed precursor Z, is a dimer of dimers containing the MoaD and MoaE proteins. The sulfur used for dithiolene formation is carried in the form of a thiocarboxylate at the MoaD C terminus. Using an intein expression system for preparation of thiocarboxylated MoaD, the mechanism of the molybdopterin synthase reaction was examined. A stoichiometry of 2 molecules of thiocarboxylated MoaD per conversion of a single precursor Z molecule to molybdopterin was observed. Examination of several synthase variants bearing mutations in the MoaE subunit identified Lys-119 as a residue essential for activity and Arg-39 and Lys-126 as other residues critical for the reaction. An intermediate of the synthase reaction was identified and characterized. This intermediate remains tightly associated with the protein and is the predominant product formed by synthase containing the K126A variant of MoaE. Mass spectral data obtained from protein-bound intermediate are consistent with a monosulfurated structure that contains a terminal phosphate group similar to that present in molybdopterin.

Structural studies of molybdopterin synthase provide insights into its catalytic mechanism.

Molybdenum cofactor biosynthesis is an evolutionarily conserved pathway present in eubacteria, archaea, and eukaryotes, including humans. Genetic deficiencies of enzymes involved in cofactor biosynthesis in humans lead to a severe and usually fatal disease. The molybdenum cofactor contains a tricyclic pyranopterin, termed molybdopterin, that bears the cis-dithiolene group responsible for molybdenum ligation. The dithiolene group of molybdopterin is generated by molybdopterin synthase, which consists of a large (MoaE) and small (MoaD) subunit. The crystal structure of molybdopterin synthase revealed a heterotetrameric enzyme in which the C terminus of each MoaD subunit is deeply inserted into a MoaE subunit to form the active site. In the activated form of the enzyme, the MoaD C terminus is present as a thiocarboxylate. The present study identified the position of the thiocarboxylate sulfur by exploiting the anomalous signal originating from the sulfur atom. The structure of molybdopterin synthase in a novel crystal form revealed a binding pocket for the terminal phosphate of molybdopterin, the product of the enzyme, and suggested a binding site for the pterin moiety present in precursor Z and molybdopterin. Finally, the crystal structure of the MoaE homodimer provides insights into the conformational changes accompanying binding of the MoaD subunit.