The molybdenum cofactor (Moco) is a redox-active prosthetic group found in the active site of Moco-dependent enzymes, which are vitally important for life. Moco biosynthesis involves several enzymes that catalyze the subsequent conversion of GTP into cyclic pyranopterin monophosphate (cPMP), molybdopterin (MPT), adenylated MPT (MPT-AMP), and finally Moco. While the underlying principles of cPMP, MPT, and MPT-AMP formation are well understood, the molybdenum insertase (Mo-insertase)-catalyzed final Moco maturation step is not. In the present study, we analyzed high-resolution X-ray datasets of the plant Mo-insertase Cnx1E that revealed two molybdate-binding sites within the active site, hence improving the current view on Cnx1E functionality. The presence of molybdate anions in either of these sites is tied to a distinctive backbone conformation, which we suggest to be essential for Mo-insertase molybdate selectivity and insertion efficiency.

The molybdenum cofactor (Moco) is found in the active site of numerous important enzymes that are critical to biological processes. The bidentate ligand that chelates molybdenum (Mo) in Moco is the pyranopterin dithiolene (molybdopterin, MPT); however, neither the mechanism of molybdate insertion into MPT nor the structure of Moco prior to its insertion into pyranopterin molybdenum enzymes is known. Here we report this final maturation step, where adenylated MPT (MPT-AMP) and molybdate are the substrates. X-ray crystallography of the Arabidopsis thaliana Mo-insertase variant Cnx1E S269D D274S identified adenylated Moco (Moco-AMP) as an unexpected intermediate in this reaction sequence. X-ray absorption spectroscopy revealed the first coordination sphere geometry of Moco trapped in the Cnx1E active site. We have used this structural information to deduce a mechanism for molybdate insertion into MPT-AMP. Given their high degree of structural and sequence similarity, we suggest that this mechanism is employed by all eukaryotic Mo-insertases.

Molybdenum insertases (Mo-insertases) catalyze the final step of molybdenum cofactor (Moco) biosynthesis, an evolutionary old and highly conserved multi-step pathway. In the first step of the pathway, GTP serves as substrate for the formation of cyclic pyranopterin monophosphate, which is subsequently converted into molybdopterin (MPT) in the second pathway step. In the following synthesis steps, MPT is adenylated yielding MPT-AMP that is subsequently used as substrate for enzyme catalyzed molybdate insertion. Molybdate insertion and MPT-AMP hydrolysis are catalyzed by the Mo-insertase E-domain. Earlier work reported a highly conserved aspartate residue to be essential for Mo-insertase functionality. In this work, we confirmed the mechanistic relevance of this residue for the Arabidopsis thaliana Mo-insertase Cnx1E. We found that the conservative substitution of Cnx1E residue Asp274 by Glu (D274E) leads to an arrest of MPT-AMP hydrolysis and hence to the accumulation of MPT-AMP. We further showed that the MPT-AMP accumulation goes in hand with the accumulation of molybdate. By crystallization and structure determination of the Cnx1E variant D274E, we identified the potential reason for the missing hydrolysis activity in the disorder of the region spanning amino acids 269 to 274. We reasoned that this is caused by the inability of a glutamate in position 274 to coordinate the octahedral Mg2+-water complex in the Cnx1E active site.

The molybdenum cofactor (Moco) is a redox active prosthetic group found in the active site of Moco dependent enzymes (Mo-enzymes). As Moco and its intermediates are highly sensitive towards oxidative damage, these are believed to be permanently protein bound during synthesis and upon maturation. As a major component of the plant Moco transfer and storage system, proteins have been identified that are capable of Moco binding and release but do not possess Moco dependent enzymatic activities. The first protein found to possess these properties was the Moco carrier protein (MCP) from the green alga Chlamydomonas reinhardtii. Here, we describe the identification and biochemical characterization of the Volvox carteri (V. carteri) MCP and, for the first time, employ a comparative analysis to elucidate the principles behind MCP Moco binding. Doing so identified a sequence region of low homology amongst the existent MCPs which we showed to be essential for Moco binding to V. carteri MCP.

The molybdenum cofactor (Moco) is a 520-Da prosthetic group that is synthesized in all domains of life. In animals, four oxidases (among them sulfite oxidase) use Moco as a prosthetic group. Moco is essential in animals; humans with mutations in genes that encode Moco biosynthetic enzymes display lethal neurological and developmental defects. Moco supplementation seems a logical therapy; however, the instability of Moco has precluded biochemical and cell biological studies of Moco transport and bioavailability. The nematode Caenorhabditis elegans can take up Moco from its bacterial diet and transport it to cells and tissues that express Moco-requiring enzymes, suggesting a system for Moco uptake and distribution. Here we show that protein-bound Moco is the stable, bioavailable species of Moco taken up by C. elegans from its diet and is an effective dietary supplement, rescuing a C. elegans model of Moco deficiency. We demonstrate that diverse Moco:protein complexes are stable and bioavailable, suggesting a new strategy for the production and delivery of therapeutically active Moco to treat human Moco deficiency.

The molybdenum cofactor (Moco) is the prosthetic group of all molybdenum-dependent enzymes except for nitrogenase. The multistep biosynthesis pathway of Moco and its function in molybdenum-dependent enzymes are already well understood. The mechanisms of Moco transfer, storage and insertion, on the other hand, are not. In the cell, Moco is usually not found in its free form and remains bound to proteins because of its sensitivity to oxidation. The green alga Chlamydomonas reinhardtii harbors a Moco carrier protein (MCP) that binds and protects Moco but is devoid of enzymatic function. It has been speculated that this MCP acts as a means of Moco storage and transport. Here, the search for potential MCPs has been extended to the prokaryotes, and many MCPs were found in cyanobacteria. A putative MCP from Rippkaea orientalis (RoMCP) was selected for recombinant production, crystallization and structure determination. RoMCP has a Rossmann-fold topology that is characteristic of nucleotide-binding proteins and a homotetrameric quaternary structure similar to that of the MCP from C. reinhardtii. In each protomer, a positively charged crevice was identified that accommodates up to three chloride ions, hinting at a potential Moco-binding site. Computational docking experiments supported this notion and gave an impression of the RoMCP–Moco complex.

The molybdenum cofactor (Moco) is a 520 dalton prosthetic group that is synthesized in a multi-step enzymatic pathway present in most Archaea, Bacteria, and Eukarya. In animals, four oxidases (among them sulfite oxidase) use Moco as a prosthetic group. Moco is essential in animals; humans with mutations in genes that encode Moco-biosynthetic enzymes display lethal neurological and developmental defects. Moco supplementation seems a logical therapy, however the instability of Moco has precluded biochemical and cell biological studies of Moco transport and bioavailability. The nematode Caenorhabditis elegans can take up Moco from its bacterial diet and transport it to cells and tissues that express Moco-requiring enzymes, suggesting a system for Moco uptake and distribution. Here we show that protein-bound Moco is the stable, bioavailable species of Moco taken up by C. elegans from its diet and is an effective dietary supplement in a C. elegans model of Moco deficiency. Diverse purified Moco:protein complexes from bacteria, bread mold, green algae, and dairy cows were able to support the growth of otherwise Moco-deficient C. elegans mutants grown on Moco-deficient E. coli. We show that these Moco:protein complexes are very stable, suggesting they may provide a strategy for the production and delivery of therapeutically active Moco to treat human Moco deficiency.

Iron-sulfur (Fe-S) clusters are essential protein cofactors. In enzymes, they are present either in the rhombic [2Fe-2S] or the cubic [4Fe-4S] form, where they are involved in catalysis and electron transfer and in the biosynthesis of metal-containing prosthetic groups like the molybdenum cofactor (Moco). Here, we give an overview of the assembly of Fe-S clusters in bacteria and humans and present their connection to the Moco biosynthesis pathway. In all organisms, Fe-S cluster assembly starts with the abstraction of sulfur from l-cysteine and its transfer to a scaffold protein. After formation, Fe-S clusters are transferred to carrier proteins that insert them into recipient apo-proteins. In eukaryotes like humans and plants, Fe-S cluster assembly takes place both in mitochondria and in the cytosol. Both Moco biosynthesis and Fe-S cluster assembly are highly conserved among all kingdoms of life. Moco is a tricyclic pterin compound with molybdenum coordinated through its unique dithiolene group. Moco biosynthesis begins in the mitochondria in a Fe-S cluster dependent step involving radical/S-adenosylmethionine (SAM) chemistry. An intermediate is transferred to the cytosol where the dithiolene group is formed, to which molybdenum is finally added. Further connections between Fe-S cluster assembly and Moco biosynthesis are discussed in detail.