Azotobacter vinelandii scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase

ABSTRACTAzotobacter vinelandii molybdenum-dependent nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires of a preexisting [Fe-S] cluster to form a [MoFe3S4] group to serve as specific donor during nitrogenase cofactor biosynthesis. Here, we show biochemical evidence for NifU being the donor of the [Fe-S] cluster. Protein-protein interaction studies using apo-NifQ and as-isolated NifU demonstrated the interaction between both proteins which is only effective when NifQ is unoccupied by its [Fe-S] cluster. The apo-NifQ iron content increased after the incubation with as-isolated NifU, reaching similar levels to holo-NifQ after the interaction between apo-NifQ and NifU with reconstituted transient [Fe4-S4] groups. These results also indicate the necessity of co-expressing NifU together with NifQ in the pathway to provide molybdenum for the biosynthesis of nitrogenase in engineered nitrogen-fixing plants.

Genome-scale modeling specifies the metabolic capabilities of Rhizophagus irregularis

Rhizophagus irregularis is one of the most extensively studied arbuscular mycorrhizal fungi (AMF) that forms symbioses with and improves the performance of many crops. Lack of transformation protocol for R. irregularis renders it challenging to investigate molecular mechanisms that shape the physiology and interactions of this AMF with plants. Here we used all published genomics, transcriptomics, and metabolomics resources to gain insights in the metabolic functionalities of R. irregularis by reconstructing its high-quality genome-scale metabolic network that considers enzyme constraints. Extensive validation tests with the enzyme-constrained metabolic model demonstrated that it can be used to: (1) accurately predict increased growth of R. irregularis on myristate with minimal medium; (2) integrate enzyme abundances and carbon source concentrations that yield growth predictions with high and significant Spearman correlation (= 0.74) to measured hyphal dry weight; and (3) simulated growth rate increases with tighter association of this AMF with the host plant across three fungal structures. Based on the validated model and system-level analyses that integrate data from transcriptomics studies, we predicted that differences in flux distributions between intraradical mycelium and arbuscles are linked to changes in amino acid and cofactor biosynthesis. Therefore, our results demonstrated that the enzyme-constrained metabolic model can be employed to pinpoint mechanisms driving developmental and physiological responses of R. irregularis to different environmental cues. In conclusion, this model can serve as a template for other AMF and paves the way to identify metabolic engineering strategies to modulate fungal metabolic traits that directly affect plant performance.

The LarB carboxylase/hydrolase forms a transient cysteinyl-pyridine intermediate during nickel-pincer nucleotide cofactor biosynthesis

Enzymes possessing the nickel-pincer nucleotide (NPN) cofactor catalyze C2 racemization or epimerization reactions of α-hydroxyacid substrates. LarB initiates synthesis of the NPN cofactor from nicotinic acid adenine dinucleotide (NaAD) by performing dual reactions: pyridinium ring C5 carboxylation and phosphoanhydride hydrolysis. Here, we show that LarB uses carbon dioxide, not bicarbonate, as the substrate for carboxylation and activates water for hydrolytic attack on the AMP-associated phosphate of C5-carboxylated-NaAD. Structural investigations show that LarB has an N-terminal domain of unique fold and a C-terminal domain homologous to aminoimidazole ribonucleotide carboxylase/mutase (PurE). Like PurE, LarB is octameric with four active sites located at subunit interfaces. The complex of LarB with NAD+, an analog of NaAD, reveals the formation of a covalent adduct between the active site Cys221 and C4 of NAD+, resulting in a boat-shaped dearomatized pyridine ring. The formation of such an intermediate with NaAD would enhance the reactivity of C5 to facilitate carboxylation. Glu180 is well positioned to abstract the C5 proton, restoring aromaticity as Cys221 is expelled. The structure of as-isolated LarB and its complexes with NAD+ and the product AMP identify additional residues potentially important for substrate binding and catalysis. In combination with these findings, the results from structure-guided mutagenesis studies lead us to propose enzymatic mechanisms for both the carboxylation and hydrolysis reactions of LarB that are distinct from that of PurE.

Pathways of iron and sulfur acquisition, cofactor assembly, destination, and storage in diverse archaeal methanogens, methanotrophs, and alkanotrophs

Archaeal methanogens, methanotrophs, and alkanotrophs have a high demand for iron (Fe) and sulfur (S); however, little is known of how they acquire, traffic, deploy, and store these elements. Here, we examined the distribution of homologs of proteins mediating key steps in Fe/S metabolism in model microorganisms, including iron(II) sensing/uptake (FeoAB), sulfide extraction from cysteine (SufS), the biosynthesis of iron-sulfur [Fe-S] clusters (SufBCDE), siroheme (Pch2-dehydrogenase), protoheme (AhbABCD), and cytochrome c (CcmCF), and iron-storage/detoxification (Bfr, FtrA, IssA), among 326 publicly available, complete or metagenome-assembled genomes of archaeal methanogens/methanotrophs/alkanotrophs. Results indicate several prevalent but non-universal features including FeoB, SufBC, and the biosynthetic apparatus for the basic tetrapyrrole scaffold as well as its siroheme (and F 430 ) derivatives. However, several early diverging genomes lacked SufS and pathways to synthesize and deploy heme. Genomes encoding complete versus incomplete heme biosynthetic pathways exhibited an equivalent prevalence of [Fe-S]-cluster binding proteins, suggesting an expansion of catalytic capabilities rather than substitution of heme for [Fe-S] in the former group. Several strains with heme binding proteins lacked heme biosynthesis capabilities while other strains with siroheme biosynthesis capability lacked homologs of known siroheme binding proteins, indicating heme auxotrophy and unknown siroheme biochemistry, respectively. While ferritin proteins involved in ferric oxide storage were widespread, those involved in storing Fe as thioferrate were unevenly distributed. Collectively, the results suggest that differences in the mechanisms of Fe and S acquisition, deployment, and storage have accompanied the diversification of methanogens/methanotrophs/alkanotrophs, possibly in response to differential availability of these elements as these organisms evolved. IMPORTANCE Archaeal methanogens, methanotrophs, and alkanotrophs, argued to be among the most ancient forms of life, have a high demand for iron (Fe) and sulfur (S) for co-factor biosynthesis, among other uses. Here, using comparative bioinformatic approaches applied to 326 genomes, we show that major differences in Fe/S acquisition, trafficking, deployment, and storage exist in this group. Variation in these characters was generally congruent with the phylogenetic placement of these genomes, indicating that variation in Fe/S usage and deployment has contributed to the diversification and ecology of these organisms. However, incongruency was observed among the distribution of cofactor biosynthesis pathways and known protein destinations for those co-factors, suggesting auxotrophy or yet to be discovered pathways for cofactor biosynthesis.

Molybdenum Enzymes and How They Support Virulence in Pathogenic Bacteria

Mononuclear molybdoenzymes are highly versatile catalysts that occur in organisms in all domains of life, where they mediate essential cellular functions such as energy generation and detoxification reactions. Molybdoenzymes are particularly abundant in bacteria, where over 50 distinct types of enzymes have been identified to date. In bacterial pathogens, all aspects of molybdoenzyme biology such as molybdate uptake, cofactor biosynthesis, and function of the enzymes themselves, have been shown to affect fitness in the host as well as virulence. Although current studies are mostly focused on a few key pathogens such as Escherichia coli, Salmonella enterica, Campylobacter jejuni, and Mycobacterium tuberculosis, some common themes for the function and adaptation of the molybdoenzymes to pathogen environmental niches are emerging. Firstly, for many of these enzymes, their role is in supporting bacterial energy generation; and the corresponding pathogen fitness and virulence defects appear to arise from a suboptimally poised metabolic network. Secondly, all substrates converted by virulence-relevant bacterial Mo enzymes belong to classes known to be generated in the host either during inflammation or as part of the host signaling network, with some enzyme groups showing adaptation to the increased conversion of such substrates. Lastly, a specific adaptation to bacterial in-host survival is an emerging link between the regulation of molybdoenzyme expression in bacterial pathogens and the presence of immune system-generated reactive oxygen species. The prevalence of molybdoenzymes in key bacterial pathogens including ESKAPE pathogens, paired with the mounting evidence of their central roles in bacterial fitness during infection, suggest that they could be important future drug targets.

A spectrum of verticality across genes

Lateral gene transfer (LGT) has impacted prokaryotic genome evolution, yet the extent to which LGT compromises vertical evolution across individual genes and individual phyla is unknown, as are the factors that govern LGT frequency across genes. Estimating LGT frequency from tree comparisons is problematic when thousands of genomes are compared, because LGT becomes difficult to distinguish from phylogenetic artefacts. Here we report quantitative estimates for verticality across all genes and genomes, leveraging a well-known property of phylogenetic inference: phylogeny works best at the tips of trees. From terminal (tip) phylum level relationships, we calculate the verticality for 19,050,992 genes from 101,422 clusters in 5,655 prokaryotic genomes and rank them by their verticality. Among functional classes, translation, followed by nucleotide and cofactor biosynthesis, and DNA replication and repair are the most vertical. The most vertically evolving lineages are those rich in ecological specialists such as Acidithiobacilli, Chlamydiae, Chlorobi and Methanococcales. Lineages most affected by LGT are the α-, β-, γ-, and δ- classes of Proteobacteria and the Firmicutes. The 2,587 eukaryotic clusters in our sample having prokaryotic homologues fail to reject eukaryotic monophyly using the likelihood ratio test. The low verticality of α-proteobacterial and cyanobacterial genomes requires only three partners—an archaeal host, a mitochondrial symbiont, and a plastid ancestor—each with mosaic chromosomes, to directly account for the prokaryotic origin of eukaryotic genes. In terms of phylogeny, the 100 most vertically evolving prokaryotic genes are neither representative nor predictive for the remaining 97% of an average genome. In search of factors that govern LGT frequency, we find a simple but natural principle: Verticality correlates strongly with gene distribution density, LGT being least likely for intruding genes that must replace a preexisting homologue in recipient chromosomes. LGT is most likely for novel genetic material, intruding genes that encounter no competing copy.

Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic Targets

Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs’ central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.