Shared functions of Fe-S cluster assembly and Moco biosynthesis.

Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In the recent years it has become evident that the availability of Fe-S clusters play an important role for the biosynthesis of Moco. First, the MoaA protein binds two [4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional [4Fe-4S] cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the L-cysteine desulfurase IscS, which is an enzyme involved in the transfer of sulfur to various acceptor proteins with a main role in the assembly of Fe-S clusters. In this review, we dissect the dependence of the production of active molybdoenzymes in detail, starting from the regulation of gene expression and further explaining sulfur delivery and Fe-S cluster insertion into target enzymes. Further, Fe-S cluster assembly is also linked to iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, we explain that the expression of the genes is dependent on an active FNR protein. FNR is a very important transcription factor that represents the master-switch for the expression of target genes in response to anaerobiosis. Moco biosynthesis is further directly dependent on the presence of ArcA and also on an active Fur protein.

The pathway for coenzyme M biosynthesis in bacteria.

Mercaptoethane sulfonate or coenzyme M (CoM) is the smallest known organic cofactor and is most commonly associated with the methane-forming step in all methanogenic archaea but is also associated with the anaerobic oxidation of methane to CO2 in anaerobic methanotrophic archaea and the oxidation of short-chain alkanes in Syntrophoarchaeum species. It has also been found in a small number of bacteria capable of the metabolism of small organics. Although many of the steps for CoM biosynthesis in methanogenic archaea have been elucidated, a complete pathway for the biosynthesis of CoM in archaea or bacteria has not been reported. Here, we present the complete CoM biosynthesis pathway in bacteria, revealing distinct chemical steps relative to CoM biosynthesis in methanogenic archaea. The existence of different pathways represents a profound instance of convergent evolution. The five-step pathway involves the addition of sulfite, the elimination of phosphate, decarboxylation, thiolation, and the reduction to affect the sequential conversion of phosphoenolpyruvate to CoM. The salient features of the pathway demonstrate reactivities for members of large aspartase/fumarase and pyridoxal 5'-phosphate-dependent enzyme families.

Sulfur amino acid metabolism and related metabotypes of autism spectrum disorder: A review of biochemical evidence for a hypothesis.

There are multiple lines of evidence for an impaired sulfur amino acid (SAA) metabolism in autism spectrum disorder (ASD). For instance, the concentrations of methionine, cysteine and S-adenosylmethionine (SAM) in body fluids of individuals with ASD is significantly lower while the concentration of S-adenosylhomocysteine (SAH) is significantly higher as compared to healthy individuals. Reduced methionine and SAM may reflect impaired remethylation pathway whereas increased SAH may reflect reduced S-adenosylhomocysteine hydrolase activity in the catabolic direction. Reduced SAM/SAH ratio reflects an impaired methylation capacity. We hypothesize multiple mechanisms to explain how the interplay of oxidative stress, neuroinflammation, mercury exposure, maternal use of valproate, altered gut microbiome and certain genetic variants may lead to these SAA metabotypes. Furthermore, we also propose a number of mechanisms to explain the metabolic consequences of abnormal SAA metabotypes. For instance in the brain, reduced SAM/SAH ratio will result in melatonin deficiency and hypomethylation of a number of biomolecules such as DNA, RNA and histones. In addition to previously proposed mechanisms, we propose that impaired activity of "radical SAM" enzymes will result in reduced endogenous lipoic acid synthesis, reduced molybdenum cofactor synthesis and impaired porphyrin metabolism leading to mitochondrial dysfunction, porphyrinuria and impaired sulfation capacity. Furthermore depletion of SAM may also lead to the disturbed mTOR signaling pathway in a subgroup of ASD. The proposed "SAM-depletion hypothesis" is an inclusive model to explain the relationship between heterogeneous risk factors and metabotypes observed in a subset of children with ASD.

Molybdenum cofactor biology, evolution and deficiency.

The molybdenum cofactor (Moco) represents an ancient metal­sulfur cofactor, which participates as catalyst in carbon, nitrogen and sulfur cycles, both on individual and global scale. Given the diversity of biological processes dependent on Moco and their evolutionary age, Moco is traced back to the last universal common ancestor (LUCA), while Moco biosynthetic genes underwent significant changes through evolution and acquired additional functions. In this review, focused on eukaryotic Moco biology, we elucidate the benefits of gene fusions on Moco biosynthesis and beyond. While originally the gene fusions were driven by biosynthetic advantages such as coordinated expression of functionally related proteins and product/substrate channeling, they also served as origin for the development of novel functions. Today, Moco biosynthetic genes are involved in a multitude of cellular processes and loss of the according gene products result in severe disorders, both related to Moco biosynthesis and secondary enzyme functions.

Human riboflavin kinase: Species-specific traits in the biosynthesis of the FMN cofactor.

Human riboflavin kinase (HsRFK) catalyzes vitamin B2 (riboflavin) phosphorylation to flavin mononucleotide (FMN), obligatory step in flavin cofactor synthesis. HsRFK expression is related to protection from oxidative stress, amyloid-ß toxicity, and some malignant cancers progression. Its downregulation alters expression profiles of clock-controlled metabolic-genes and destroys flavins protection on stroke treatments, while its activity reduction links to protein-energy malnutrition and thyroid hormones decrease. We explored specific features of the mechanisms underlying the regulation of HsRFK activity, showing that both reaction products regulate it through competitive inhibition. Fast-kinetic studies show that despite HsRFK binds faster and preferably the reaction substrates, the complex holding both products is kinetically most stable. An intricate ligand binding landscape with all combinations of substrates/products competing with the catalytic complex and exhibiting moderate cooperativity is also presented. These data might contribute to better understanding the molecular bases of pathologies coursing with aberrant HsRFK availability, and envisage that interaction with its client-apoproteins might favor FMN release. Finally, HsRFK parameters differ from those of the so far evaluated bacterial counterparts, reinforcing the idea of species-specific mechanisms in RFK catalysis. These observations support HsRFK as potential therapeutic target because of its key functions, while also envisage bacterial RFK modules as potential antimicrobial targets.

Iron-Dependent Regulation of Molybdenum Cofactor Biosynthesis Genes in Escherichia coli.

Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In recent years it has become obvious that the availability of iron plays an important role in the biosynthesis of Moco. First, the MoaA protein binds two [4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional [4Fe-4S] cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the l-cysteine desulfurase IscS, which is a shared protein with a main role in the assembly of Fe-S clusters. In this report, we investigated the transcriptional regulation of the moaABCDE operon by focusing on its dependence on cellular iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, our data show that the regulation of the moaABCDE operon at the level of transcription is only marginally influenced by the availability of iron. Nevertheless, intracellular levels of Moco were decreased under iron-limiting conditions, likely based on an inactive MoaA protein in addition to lower levels of the l-cysteine desulfurase IscS, which simultaneously reduces the sulfur availability for Moco production.IMPORTANCE FNR is a very important transcriptional factor that represents the master switch for the expression of target genes in response to anaerobiosis. Among the FNR-regulated operons in Escherichia coli is the moaABCDE operon, involved in Moco biosynthesis. Molybdoenzymes have essential roles in eukaryotic and prokaryotic organisms. In bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. This work investigates the connection of iron availability to the biosynthesis of Moco and the production of active molybdoenzymes.

The peacefulness gene promotes aggression in Drosophila.

Natural aggressiveness is commonly observed in all animal species, and is displayed frequently when animals compete for food, territory and mating. Aggression is an innate behaviour, and is influenced by both environmental and genetic factors. However, the genetics of aggression remains largely unclear. In this study, we identify the peacefulness (pfs) gene as a novel player in the control of male-male aggression in Drosophila. Mutations in pfs decreased intermale aggressiveness, but did not affect locomotor activity, olfactory avoidance response and sexual behaviours. pfs encodes for the evolutionarily conserved molybdenum cofactor (MoCo) synthesis 1 protein (Mocs1), which catalyzes the first step in the MoCo biosynthesis pathway. Neuronal-specific knockdown of pfs decreased aggressiveness. By contrast, overexpression of pfs greatly increased aggressiveness. Knocking down Cinnamon (Cin) catalyzing the final step in the MoCo synthesis pathway, caused a pfs-like aggression phenotype. In humans, inhibition of MoCo-dependent enzymes displays anti-aggressive effects. Thus, the control of aggression by Pfs-dependent MoCo pathways may be conserved throughout evolution.

Lessons From the Studies of a CC Bond Forming Radical SAM Enzyme in Molybdenum Cofactor Biosynthesis.

MoaA is one of the founding members of the radical S-adenosyl-L-methionine (SAM) superfamily, and together with the second enzyme, MoaC, catalyzes the construction of the pyranopterin backbone structure of the molybdenum cofactor (Moco). However, the exact functions of both MoaA and MoaC had remained ambiguous for more than 2 decades. Recently, their functions were finally elucidated through successful characterization of the MoaA product as 3',8-cyclo-7,8-dihydro-GTP (3',8-cH2GTP), which was shown to be converted to cyclic pyranopterin monophosphate (cPMP) by MoaC. 3',8-cH2GTP was produced in a small quantity and was highly oxygen sensitive, which explains why this compound had previously eluded characterization. This chapter describes the methodologies for the characterization of MoaA, MoaC, and 3',8-cH2GTP, which together significantly altered the view of the mechanism of the pyranopterin backbone construction during the Moco biosynthesis. Through this chapter, we hope to share not only the protocols to study the first step of Moco biosynthesis but also the lessons we learned from the characterization of the chemically labile biosynthetic intermediate, which would be informative for the study of many other metabolic pathways and enzymes.

Diversity of structures, catalytic mechanisms and processes of cofactor biosynthesis of tryptophylquinone-bearing enzymes.

Tryptophyquinone-bearing enzymes contain protein-derived cofactors formed by posttranslational modifications of Trp residues. Tryptophan tryptophylquinone (TTQ) is comprised of a di-oxygenated Trp residue, which is cross-linked to another Trp residue. Cysteine tryptophylquinone (CTQ) is comprised of a di-oxygenated Trp residue, which is cross-linked to a Cys residue. Despite the similarity of these cofactors, it has become evident in recent years that the overall structures of the enzymes that possess these cofactors vary, and that the gene clusters that encode the enzymes are quite diverse. While it had been long assumed that all tryptophylquinone enzymes were dehydrogenases, recently discovered classes of these enzymes are oxidases. A common feature of enzymes that have these cofactors is that the posttranslational modifications that form the mature cofactors are catalyzed by a modifying enzyme. However, it is now clear that modifying enzymes are different for different tryptophylquinone enzymes. For methylamine dehydrogenase a di-heme enzyme, MauG, is needed to catalyze TTQ biosynthesis. However, no gene similar to mauG is present in the gene clusters that encode the other enzymes, and the recently characterized family of CTQ-dependent oxidases, termed LodA-like proteins, require a flavoenzyme for cofactor biosynthesis.

C-C bond forming radical SAM enzymes involved in the construction of carbon skeletons of cofactors and natural products.

Covering: up to the end of 2017 C-C bond formations are frequently the key steps in cofactor and natural product biosynthesis. Historically, C-C bond formations were thought to proceed by two electron mechanisms, represented by Claisen condensation in fatty acids and polyketide biosynthesis. These types of mechanisms require activated substrates to create a nucleophile and an electrophile. More recently, increasing number of C-C bond formations catalyzed by radical SAM enzymes are being identified. These free radical mediated reactions can proceed between almost any sp3 and sp2 carbon centers, allowing introduction of C-C bonds at unconventional positions in metabolites. Therefore, free radical mediated C-C bond formations are frequently found in the construction of structurally unique and complex metabolites. This review discusses our current understanding of the functions and mechanisms of C-C bond forming radical SAM enzymes and highlights their important roles in the biosynthesis of structurally complex, naturally occurring organic molecules. Mechanistic consideration of C-C bond formation by radical SAM enzymes identifies the significance of three key mechanistic factors: radical initiation, acceptor substrate activation and radical quenching. Understanding the functions and mechanisms of these characteristic enzymes will be important not only in promoting our understanding of radical SAM enzymes, but also for understanding natural product and cofactor biosynthesis.

Radical Breakthroughs in Natural Product and Cofactor Biosynthesis.

The radical SAM (S-adenosyl-l-methionine) superfamily is one of the largest group of enzymes with >113000 annotated sequences [Landgraf, B. J., et al. (2016) Annu. Rev. Biochem. 85, 485-514]. Members of this superfamily catalyze the reductive cleavage of SAM using an oxygen sensitive 4Fe-4S cluster to transiently generate 5'-deoxyadenosyl radical that is subsequently used to initiate diverse free radical-mediated reactions. Because of the unique reactivity of free radicals, radical SAM enzymes frequently catalyze chemically challenging reactions critical for the biosynthesis of unique structures of cofactors and natural products. In this Perspective, I will discuss the impact of characterizing novel functions in radical SAM enzymes on our understanding of biosynthetic pathways and use two recent examples from my own group with a particular emphasis on two radical SAM enzymes that are responsible for carbon skeleton formation during the biosynthesis of a cofactor and natural products.

Horizontal acquisition of a hypoxia-responsive molybdenum cofactor biosynthesis pathway contributed to Mycobacterium tuberculosis pathoadaptation.

The unique ability of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, to persist for long periods of time in lung hypoxic lesions chiefly contributes to the global burden of latent TB. We and others previously reported that the M. tuberculosis ancestor underwent massive episodes of horizontal gene transfer (HGT), mostly from environmental species. Here, we sought to explore whether such ancient HGT played a part in M. tuberculosis evolution towards pathogenicity. We were interested by a HGT-acquired M. tuberculosis-specific gene set, namely moaA1-D1, which is involved in the biosynthesis of the molybdenum cofactor. Horizontal acquisition of this gene set was striking because homologues of these moa genes are present all across the Mycobacterium genus, including in M. tuberculosis. Here, we discovered that, unlike their paralogues, the moaA1-D1 genes are strongly induced under hypoxia. In vitro, a M. tuberculosis moaA1-D1-null mutant has an impaired ability to respire nitrate, to enter dormancy and to survive in oxygen-limiting conditions. Conversely, heterologous expression of moaA1-D1 in the phylogenetically closest non-TB mycobacterium, Mycobacterium kansasii, which lacks these genes, improves its capacity to respire nitrate and grants it with a marked ability to survive oxygen depletion. In vivo, the M. tuberculosis moaA1-D1-null mutant shows impaired survival in hypoxic granulomas in C3HeB/FeJ mice, but not in normoxic lesions in C57BL/6 animals. Collectively, our results identify a novel pathway required for M. tuberculosis resistance to host-imposed stress, namely hypoxia, and provide evidence that ancient HGT bolstered M. tuberculosis evolution from an environmental species towards a pervasive human-adapted pathogen.

Functional Complementation Studies Reveal Different Interaction Partners of Escherichia coli IscS and Human NFS1.

The trafficking and delivery of sulfur to cofactors and nucleosides is a highly regulated and conserved process among all organisms. All sulfur transfer pathways generally have an l-cysteine desulfurase as an initial sulfur-mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of sulfur-containing biomolecules like iron-sulfur (Fe-S) clusters, thiamine, biotin, lipoic acid, the molybdenum cofactor (Moco), and thiolated nucleosides in tRNA. The human l-cysteine desulfurase NFS1 and the Escherichia coli homologue IscS share a level of amino acid sequence identity of ∼60%. While E. coli IscS has a versatile role in the cell and was shown to have numerous interaction partners, NFS1 is mainly localized in mitochondria with a crucial role in the biosynthesis of Fe-S clusters. Additionally, NFS1 is also located in smaller amounts in the cytosol with a role in Moco biosynthesis and mcm5s2U34 thio modifications of nucleosides in tRNA. NFS1 and IscS were conclusively shown to have different interaction partners in their respective organisms. Here, we used functional complementation studies of an E. coli iscS deletion strain with human NFS1 to dissect their conserved roles in the transfer of sulfur to a specific target protein. Our results show that human NFS1 and E. coli IscS share conserved binding sites for proteins involved in Fe-S cluster assembly like IscU, but not with proteins for tRNA thio modifications or Moco biosynthesis. In addition, we show that human NFS1 was almost fully able to complement the role of IscS in Moco biosynthesis when its specific interaction partner protein MOCS3 from humans was also present.

Whole transcriptomic and proteomic analyses of an isogenic M. tuberculosis clinical strain with a naturally occurring 15 Kb genomic deletion.

Tuberculosis remains one of the most difficult to control infectious diseases in the world. Many different factors contribute to the complexity of this disease. These include the ability of the host to control the infection which may directly relate to nutritional status, presence of co-morbidities and genetic predisposition. Pathogen factors, in particular the ability of different Mycobacterium tuberculosis strains to respond to the harsh environment of the host granuloma, which includes low oxygen and nutrient availability and the presence of damaging radical oxygen and nitrogen species, also play an important role in the success of different strains to cause disease. In this study we evaluated the impact of a naturally occurring 12 gene 15 Kb genomic deletion on the physiology and virulence of M. tuberculosis. The strains denominated ON-A WT (wild type) and ON-A NM (natural mutant) were isolated from a previously reported TB outbreak in an inner city under-housed population in Toronto, Canada. Here we subjected these isogenic strains to transcriptomic (via RNA-seq) and proteomic analyses and identified several gene clusters with differential expression in the natural mutant, including the DosR regulon and the molybdenum cofactor biosynthesis genes, both of which were found in lower abundance in the natural mutant. We also demonstrated lesser virulence of the natural mutant in the guinea pig animal model. Overall, our findings suggest that the ON-A natural mutant is less fit to cause disease, but nevertheless has the potential to cause extended transmission in at-risk populations.

Iron assimilation and utilization in anaerobic ammonium oxidizing bacteria.

The most abundant transition metal in biological systems is iron. It is incorporated into protein cofactors and serves either catalytic, redox or regulatory purposes. Anaerobic ammonium oxidizing (anammox) bacteria rely heavily on iron-containing proteins - especially cytochromes - for their energy conservation, which occurs within a unique organelle, the anammoxosome. Both their anaerobic lifestyle and the presence of an additional cellular compartment challenge our understanding of iron processing. Here, we combine existing concepts of iron uptake, utilization and metabolism, and cellular fate with genomic and still limited biochemical and physiological data on anammox bacteria to propose pathways these bacteria may employ.

The Role of SufS Is Restricted to Fe-S Cluster Biosynthesis in Escherichia coli.

In Escherichia coli, two different systems that are important for the coordinate formation of Fe-S clusters have been identified, namely, the ISC and SUF systems. The ISC system is the housekeeping Fe-S machinery, which provides Fe-S clusters for numerous cellular proteins. The IscS protein of this system was additionally revealed to be the primary sulfur donor for several sulfur-containing molecules with important biological functions, among which are the molybdenum cofactor (Moco) and thiolated nucleosides in tRNA. Here, we show that deletion of central components of the ISC system in addition to IscS leads to an overall decrease in Fe-S cluster enzyme and molybdoenzyme activity in addition to a decrease in the number of Fe-S-dependent thiomodifications of tRNA, based on the fact that some proteins involved in Moco biosynthesis and tRNA thiolation are Fe-S-dependent. Complementation of the ISC deficient strains with the suf operon restored the activity of Fe-S-containing proteins, including the MoaA protein, which is involved in the conversion of 5'GTP to cyclic pyranopterin monophosphate in the fist step of Moco biosynthesis. While both systems share a high degree of similarity, we show that the function of their respective l-cysteine desulfurase IscS or SufS is specific for each cellular pathway. It is revealed that SufS cannot play the role of IscS in sulfur transfer for the formation of 2-thiouridine, 4-thiouridine, or the dithiolene group of molybdopterin, being unable to interact with TusA or ThiI. The results demonstrate that the role of the SUF system is exclusively restricted to Fe-S cluster assembly in the cell.

Structure of the Reduced Copper Active Site in Preprocessed Galactose Oxidase: Ligand Tuning for One-Electron O2 Activation in Cofactor Biogenesis.

Galactose oxidase (GO) is a copper-dependent enzyme that accomplishes 2e- substrate oxidation by pairing a single copper with an unusual cysteinylated tyrosine (Cys-Tyr) redox cofactor. Previous studies have demonstrated that the post-translational biogenesis of Cys-Tyr is copper- and O2-dependent, resulting in a self-processing enzyme system. To investigate the mechanism of cofactor biogenesis in GO, the active-site structure of Cu(I)-loaded GO was determined using X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy, and density-functional theory (DFT) calculations were performed on this model. Our results show that the active-site tyrosine lowers the Cu potential to enable the thermodynamically unfavorable 1e- reduction of O2, and the resulting Cu(II)-O2•- is activated toward H atom abstraction from cysteine. The final step of biogenesis is a concerted reaction involving coordinated Tyr ring deprotonation where Cu(II) coordination enables formation of the Cys-Tyr cross-link. These spectroscopic and computational results highlight the role of the Cu(I) in enabling O2 activation by 1e- and the role of the resulting Cu(II) in enabling substrate activation for biogenesis.

Mouse model for molybdenum cofactor deficiency type B recapitulates the phenotype observed in molybdenum cofactor deficient patients.

Molybdenum cofactor (MoCo) deficiency is a rare, autosomal-recessive disorder, mainly caused by mutations in MOCS1 (MoCo deficiency type A) or MOCS2 (MoCo deficiency type B) genes; the absence of active MoCo results in a deficiency in all MoCo-dependent enzymes. Patients with MoCo deficiency present with neonatal seizures, feeding difficulties, severe developmental delay, brain atrophy and early childhood death. Although substitution therapy with cyclic pyranopterin monophosphate (cPMP) has been successfully used in both Mocs1 knockout mice and in patients with MoCo deficiency type A, there is currently no Mocs2 knockout mouse and no curative therapy for patients with MoCo deficiency type B. Therefore, we generated and characterized a Mocs2-null mouse model of MoCo deficiency type B. Expression analyses of Mocs2 revealed a ubiquitous expression pattern; however, at the cellular level, specific cells show prominent Mocs2 expression, e.g., neuronal cells in cortex, hippocampus and brainstem. Phenotypic analyses demonstrated that Mocs2 knockout mice failed to thrive and died within 11 days after birth. None of the tested MoCo-dependent enzymes were active in Mocs2-deficient mice, leading to elevated concentrations of purines, such as hypoxanthine and xanthine, and non-detectable levels of uric acid in the serum and urine. Moreover, elevated concentrations of S-sulfocysteine were measured in the serum and urine. Increased levels of xanthine resulted in bladder and kidney stone formation, whereas increased concentrations of toxic sulfite triggered neuronal apoptosis. In conclusion, Mocs2-deficient mice recapitulate the severe phenotype observed in humans and can now serve as a model for preclinical therapeutic approaches for MoCo deficiency type B.

Structural Framework for Metal Incorporation during Molybdenum Cofactor Biosynthesis.

The molybdenum cofactor (Moco) is essential for the catalytic activity of all molybdenum-containing enzymes with the exception of nitrogenase. Moco biosynthesis follows an evolutionarily highly conserved pathway and genetic deficiencies in the corresponding human enzymes result in Moco deficiency, which manifests itself in severe neurological symptoms and death in childhood. In humans the final steps of Moco biosynthesis are catalyzed by gephyrin, specifically the penultimate adenylation of molybdopterin (MPT) by its N-terminal G domain (GephG) and the final metal incorporation by its C-terminal E domain (GephE). To better understand the poorly defined molecular framework of this final step, we determined high-resolution crystal structures of GephE in the apo state and in complex with ADP, AMP, and molybdate. Our data provide novel insights into the catalytic steps leading to final Moco maturation, namely deadenylation as well as molybdate binding and insertion.

Mechanistic Investigation of cPMP Synthase in Molybdenum Cofactor Biosynthesis Using an Uncleavable Substrate Analogue.

Molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During its biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin monophosphate (cPMP) through the action of two enzymes, MoaA and MoaC. Recent studies revealed that MoaC catalyzes the majority of the transformation and produces cPMP from a unique cyclic nucleotide, 3',8-cyclo-7,8-dihydro-GTP (3',8-cH2GTP). However, the mechanism by which MoaC catalyzes this complex rearrangement is largely unexplored. Here, we report the mechanistic characterization of MoaC using an uncleavable substrate analogue, 3',8-cH2GMP[CH2]PP, as a probe to investigate the timing of cyclic phosphate formation. Using partially active MoaC variants, 3',8-cH2GMP[CH2]PP was found to be accepted by MoaC as a substrate and was converted to an analogue of the previously described MoaC reaction intermediate, suggesting that the early stage of catalysis proceeds without cyclic phosphate formation. In contrast, when it was incubated with wt-MoaC, 3',8-cH2GMP[CH2]PP caused mechanism-based inhibition. Detailed characterization of the inhibited MoaC suggested that 3',8-cH2GMP[CH2]PP is mainly converted to a molecule (compound Y) with an acid-labile triaminopyrimidinone base without an established pyranopterin structure. MS analysis of MoaC treated with 3',8-cH2GMP[CH2]PP provided strong evidence that compound Y forms a tight complex with MoaC likely through a covalent linkage. These observations are consistent with a mechanism in which cyclic phosphate ring formation proceeds in concert with the pterin ring formation. This mechanism would provide a thermodynamic driving force to complete the formation of the unique tetracyclic structure of cPMP.