Mo and W are found in the mononuclear form in the active sites of diverse enzymes in all three domains of life.46–48
The active sites of these enzymes include the metal ion coordinated to pyranopterin molecules and to a variable number of other ligands, such as oxygen, sulfur and selenium.49, 50
In addition, these proteins may have other redox cofactors, such as iron–sulfur centers, flavins and hemes, which are involved in intramolecular and intermolecular electron transfer processes.49
Much effort has been made on identifying and characterizing Moco biosynthesis components and Mo-dependent enzymes in various organisms. In contrast, occurrence and evolution of the overall Mo utilization trait remained unclear. In this study, we analyzed phylogenetic profiles and regulation of Mo uptake systems, Moco biosynthesis genes and Mo-containing proteins to better understand evolution and current use of Mo in nature. Our data reveal patterns and properties of Mo utilization among organisms with sequenced genomes and provide new insights into understanding the dynamic evolution of the Mo utilization trait in prokaryotes and eukaryotes.
The widespread distribution of the Mo utilization trait in prokaryotes suggests that this trace element could be used by essentially all prokaryotic phyla. In contrast, the absence of the Mo utilization trait from several evolutionarily distant phyla (e. g., Firmicutes/Mollicutes and Chlamydiae) implied a loss of this trait from these clades. There was a very good correspondence between the occurrence of the Mo biosynthesis pathway and the presence of known Moco-containing protein families. However, a few exceptions wherein some organisms lacked either Moco-containing proteins or Moco biosynthesis components suggest the presence of additional Moco-dependent protein families or alternative Mo utilization pathways in these organisms.
Besides the classic ModABC transport system, a distant ModABC-like group was predicted in Pyrobaculum. Although the ModA-like proteins appeared to be an outgroup of all three known Mo/W transporters, they belong to the same COG as E. coli ModA. The presence of modB-like and modC-like genes (as well as the modD gene) in the same operon implied that they form a distant group of ModABC transporters and are involved in Mo/W uptake. Orthologs of this group could be found only in Pyrobaculum species but not in other sister species in the same archaeal phylum. It is possible that these ModABC-like transporters evolved from an ancestral ModABC system and diverged rapidly in Pyrobaculum. On the other hand, MOT1, which is the only known Mo transporter in eukaryotes, was detected only in one-third of Mo-utilizing organisms, suggesting that most eukaryotes (including all animals) use additional unknown transport system(s) for Mo uptake.
We investigated ModE-related ModABC regulation in prokaryotes. Surprisingly, less than 30% of Mo-utilizing organisms possessed full-length ModE regulators. Over 70% bacteria and 80% archaea appeared not to use E. coli-type ModE for ModABC regulation. Orphan Mop or Di-Mop proteins are not specific for ModE-related regulation because they occur also in other proteins with distinct functions (e.g., the Mop domain is present in the C-terminus of ModC, and Di-Mop domain is present in ModG which is implicated in intracellular Mo homeostasis). Although some species contain either both ModE_N and Mop/Di-mop proteins (which suggests a function similar to that of ModE) or orphan ModE_N (which may mediate weak ModABC regulation), almost half of ModABC-containing organisms lacked ModE-type ModABC regulation. This finding suggests the presence of novel or unspecific pathways for molybdate uptake in these organisms. In addition, the occurrence of different fusion proteins composed of ModE_N and Mop domains suggests the presence of more complex regulatory networks for Mo uptake, and Moco biosynthesis and utilization. Analysis of the gene neighborhoods of ModE_N/ModE and TupABC/WtpABC transporters implied that the two secondary Mo/W transporters may be also regulated by ModE-type system in some organisms.
Analysis of Mo-containing proteins provided a straightforward approach to analyze the distribution and evolution of molybdoproteomes in various organisms. AOR was the first enzyme that was structurally characterized as a protein containing a Moco-type cofactor, and it has been proposed to be the primary enzyme responsible for the interconversion of aldehydes and carboxylates in archaea.51
However, it is the rarest known bacterial Moco-containing protein, suggesting that AOR-dependent oxidation of aldehydes is not needed for most bacterial species. The other three molybdoenzyme families are distributed much more widely, especially the DMSOR family, which is found in almost all Mo-utilizing bacteria and all Mo-utilizing archaea. Enzymes of the DMSOR family catalyze a variety of reactions that involve oxygen atom transfer to or from an available electron pair of a substrate or cleavage of a C-H bond.2, 10, 52–55
NR (dissimilatory) and formate dehydrogenase are the two major members of the DMSOR family. The formate dehydrogenase alpha subunit (FdhA) is also a selenocysteine (Sec)-containing protein that may be responsible for maintaining the Sec-decoding trait in prokaryotes.56
We compared the distribution of Mo- and Sec-utilizing organisms in both prokaryotes and eukaryotes, and found that Sec-utilizing organisms were essentially a subset of Moco-dependent organisms in prokaryotes (, Table S1
). These data suggest that the Sec trait is dependent on the Mo utilization trait in prokaryotes because of the function of formate dehydrogenase, which is a widespread Mo-enzyme and the main user of Se in prokaryotes. In addition, occurrence of the only non-Moco-containing protein, nitrogenase, was limited in both bacteria and archaea. This enzyme is used by several organisms to fix atmospheric nitrogen gas (N2
). The fact that it was found in all methanogenic archaea implied that the function of this protein is essential for these organisms.
Distribution of Moco utilization and Sec utilization in the three domains of life
We attempted to generate a general evolutionary model of Mo utilization in the three domains of life. The Moco biosynthesis pathway and at least two molybdoenzyme families (SO and XO) were likely present in the last universal common ancestor. The Moco utilization trait is evolutionarily conserved in most prokaryotic and eukaryotic species due to the important redox reactions catalyzed by molybdoenzymes in carbon, nitrogen and sulfur metabolism. In addition, an independent loss of the Moco utilization trait (instead of an HGT from other species) and perhaps an appearance of alternative Mo-independent pathways has a role in the evolution of Mo utilization.
We hypothesized that, since both the Moco biosynthesis trait and molybdoenzymes were present (or both were absent) in organisms, and these patterns were observed in various bacterial phyla, certain common factors (e.g., habitat) may have affected the acquisition/loss of Mo utilization. To examine this possibility, we analyzed a role of environmental conditions (e.g., habitat, oxygen requirement, optimal temperature and optimal pH) and other factors (e.g., genome size, G+C content) in Mo utilization in sequenced prokaryotes. Previously, a similar strategy was used to analyze the evolution of Se in bacteria.56
shows the distribution of organisms that possess or lack Moco utilization with respect to several such factors.
Relationship between environmental factors, properties of organisms and the Mo utilization trait
We found that the majority of bacteria that do not utilize Moco were host-associated (i.e., parasites or symbionts, ), implying that host-associated lifestyle often leads to the loss of Mo utilization, perhaps due to limited space and resources or availability of Mo pathways of the host. This is consistent with the observation in Firmicutes/Mollicutes and Chlamydiae, all of which are host-associated and could not utilize Moco. This idea is also supported by analysis of Mo utilization in Alphaproteobacteria/Rickettsiales. In this phylum, only one out of 19 organisms utilized Mo (Candidatus Pelagibacter ubique, a marine bacterium living in ocean surface water). However, it is also the only non-host-associated organism in this clade.
Our data suggested a complete loss of the Mo utilization trait in all host-related organisms in this phylum instead of an HGT into Candidatus Pelagibacter ubique. In addition, in many phyla, genomes of Moco-utilizing organisms had a significantly higher G+C content, suggesting that the increase in G+C content correlates with increased Mo utilization (). Organisms with low G+C content (i.e., GC <40%) that lack the Moco utilization trait were were found in a variety of phyla, indicating that such correlation is significant. The reason why low G+C content organisms in different clades lost the Moco utilization trait is not clear. Other factors, such as oxygen requirement, gram strain, optimal temperature and pH, did not appear to have a role in Mo utilization. In archaea, only two organisms, Methanosphaera stadtmanae (the only sequenced parasite in archaea) and Nanoarchaeum equitans (an ancient hyperthermophilic and anaerobic obligate symbiont with a small genome that has lost the ability to use most trace elements such as nickel, cobalt, copper and selenium), lacked Mo utilization and both genomes had a very low G+C content (27.6% and 31.6%). These data provided additional support for our observation in bacteria. Thus, host-associated lifestyle as well as reduced G+C content seem to correlate with the loss of Mo utilization.
We also examined the distribution, based on the factors discussed above, of different molybdoenzyme families, and similar trends were found. Moreover, additional features were observed for different molybdoenzymes (). For example, organisms possessing AOR proteins favor an anaerobic environment, whereas organisms containing SO, XO or DMSOR proteins favor aerobic conditions. Organisms containing nitrogenase favor both anaerobic and relatively warm conditions (no psychrophilic organism possessed nitrogenase). These data illustrate that, although being dependent on the same processes, such as Mo availability and Moco synthesis, different Mo enzymes are subject to independent and dynamic evolutionary processes.
Relationship between environmental factors, properties of organisms and different molybdoenzymes
Similar investigation of molybdoenzymes in eukaryotes provided the information on Mo utilization in this domain of life. As in prokaryotes, distribution of eukaryotic Mo-containing proteins essentially matched the Moco utilization trait. However, only SO (including NR and SO) and XO (including XDH and AO) families could be detected, suggesting a much smaller molybdoproteome in eukaryotes than in prokaryotes. The functional roles of these four subfamilies have been investigated in different organisms.2, 3, 7
Besides NR, which is a key enzyme of nitrate assimilation and does not occur in animals, the other three enzymes are present in a variety of clades including unicellular organisms and animals. SO catalyzes the oxidation of sulfite to sulfate (the final step in the degradation of sulfur-containing amino acids).7
XDH is a key enzyme in purine degradation that oxidizes both hypoxanthine to xanthine and xanthine to uric acid, whereas AO catalyzes the oxidation of a variety of aromatic and nonaromatic heterocycles and aldehydes and converts them to the respective carboxylic acids.7
All parasites lost the ability to synthesize Moco, which is consistent with what we found in prokaryotes, suggesting that Mo utilization may have been present in the eukaryotic progenitor and became unnecessary for parasites because of the reduced availability of Mo or dependence on the corresponding metabolic pathways of the host. Both Mo-dependent and Mo-independent organisms were found among fungi. The recent loss of the Mo biosynthesis pathway and Mo-dependent NR in most yeasts, including S. cerevisiae
, suggested that Mo-dependent nitrate assimilation may be unnecessary or have been replaced by other pathways in these organisms. It is known that nitrate assimilation is one of two major biological processes by which inorganic nitrogen is converted to ammonia and hence to organic nitrogen.58
Although S. cerevisiae
lacks both Moco biosynthesis trait and NR, it contains a number of genes which convert glutamine to glutamate, providing a major source of organic nitrogen.59
In addition, glutathione (GSH) stored in the yeast vacuole can serve as an alternative nitrogen source during nitrogen starvation.60
It is unclear whether the ancestor of yeasts possessed other Mo-binding enzymes. However, alternative Mo-independent pathways for sulfur and carbon metabolism may have evolved in yeasts. Both Mo-dependent and Mo-independent fungi are free-living organisms and in this case we could not identify a common environmental factor related to Mo utilization. Thus, additional unidentified factors may have affected Mo utilization in fungi. A future challenge would be to discover these factors as well as additional features influencing Mo utilization in the three domains of life.
In conclusion, we report a comprehensive comparative genomics analysis of Mo utilization in prokaryotes and eukaryotes by examining occurrence of proteins involved in Moco biosynthesis, Mo transport and Mo utilization (molybdoenzymes). Our data reveal a complex and dynamic evolutionary process of Mo utilization. Most bacteria and archaea utilize Mo, with the exception of parasites and organisms with low genomic G+C content. A distant group of ModABC transport system was identified in Pyrobaculum species. Regulation of Mo uptake must be more complex than previously thought, as ModE-type ModABC regulatory systems occurred in only a limited number of Moco-utilizing organisms. In contrast to the wide use of Mo in prokaryotes, the utilization of this element in eukaryotes is more restricted, both with regard to the number of organisms that depend on Mo and the number of molybdoprotein families that occur in them. Again, host-associated conditions appear to lead to the loss of Mo utilization.