From dynamin related proteins structures and oligomers to membrane fusion mediated by mitofusins.

Mitochondria assemble in a highly dynamic network where interconnected tubules evolve in length and size through regulated cycles of fission and fusion of mitochondrial membranes thereby adapting to cellular needs. Mitochondrial fusion and fission processes are mediated by specific sets of mechano-chemical large GTPases that belong to the Dynamin-Related Proteins (DRPs) super family. DRPs bind to cognate membranes and auto-oligomerize to drive lipid bilayers remodeling in a nucleotide dependent manner. Although structural characterization and mechanisms of DRPs that mediate membrane fission are well established, the capacity of DRPs to mediate membrane fusion is only emerging. In this review, we discuss the distinct structures and mechanisms of DRPs that trigger the anchoring and fusion of biological membranes with a specific focus on mitofusins that are dedicated to the fusion of mitochondrial outer membranes. In particular, we will highlight oligomeric assemblies of distinct DRPs and confront their mode of action against existing models of mitofusins assemblies with emphasis on recent biochemical, structural and computational reports. As we will see, the literature brings valuable insights into the presumed macro-assemblies mitofusins may form during anchoring and fusion of mitochondrial outer membranes.

Identification of Modulators of the C. elegans Aryl Hydrocarbon Receptor and Characterization of Transcriptomic and Metabolic AhR-1 Profiles.

The Aryl hydrocarbon Receptor (AhR) is a xenobiotic sensor in vertebrates, regulating the metabolism of its own ligands. However, no ligand has been identified to date for any AhR in invertebrates. In C. elegans, the AhR ortholog, AHR-1, displays physiological functions. Therefore, we compared the transcriptomic and metabolic profiles of worms expressing AHR-1 or not and investigated the putative panel of chemical AHR-1 modulators. The metabolomic profiling indicated a role for AHR-1 in amino acids, carbohydrates, and fatty acids metabolism. The transcriptional profiling in neurons expressing AHR-1, identified 95 down-regulated genes and 76 up-regulated genes associated with neuronal and metabolic functions in the nervous system. A gene reporter system allowed us to identify several AHR-1 modulators including bacterial, dietary, or environmental compounds. These results shed new light on the biological functions of AHR-1 in C. elegans and perspectives on the evolution of the AhR functions across species.

Structural basis for substrate selectivity and nucleophilic substitution mechanisms in human adenine phosphoribosyltransferase catalyzed reaction.

The reversible adenine phosphoribosyltransferase enzyme (APRT) is essential for purine homeostasis in prokaryotes and eukaryotes. In humans, APRT (hAPRT) is the only enzyme known to produce AMP in cells from dietary adenine. APRT can also process adenine analogs, which are involved in plant development or neuronal homeostasis. However, the molecular mechanism underlying substrate specificity of APRT and catalysis in both directions of the reaction remains poorly understood. Here we present the crystal structures of hAPRT complexed to three cellular nucleotide analogs (hypoxanthine, IMP, and GMP) that we compare with the phosphate-bound enzyme. We established that binding to hAPRT is substrate shape-specific in the forward reaction, whereas it is base-specific in the reverse reaction. Furthermore, a quantum mechanics/molecular mechanics (QM/MM) analysis suggests that the forward reaction is mainly a nucleophilic substitution of type 2 (SN2) with a mix of SN1-type molecular mechanism. Based on our structural analysis, a magnesium-assisted SN2-type mechanism would be involved in the reverse reaction. These results provide a framework for understanding the molecular mechanism and substrate discrimination in both directions by APRTs. This knowledge can play an instrumental role in the design of inhibitors, such as antiparasitic agents, or adenine-based substrates.

Structural Insights into the Forward and Reverse Enzymatic Reactions in Human Adenine Phosphoribosyltransferase.

Phosphoribosyltransferases catalyze the displacement of a PRPP α-1'-pyrophosphate to a nitrogen-containing nucleobase. How they control the balance of substrates/products binding and activities is poorly understood. Here, we investigated the human adenine phosphoribosyltransferase (hAPRT) that produces AMP in the purine salvage pathway. We show that a single oxygen atom from the Tyr105 side chain is responsible for selecting the active conformation of the 12 amino acid long catalytic loop. Using in vitro, cellular, and in crystallo approaches, we demonstrated that Tyr105 is key for the fine-tuning of the kinetic activity efficiencies of the forward and reverse reactions. Together, our results reveal an evolutionary pressure on the strictly conserved Tyr105 and on the dynamic motion of the flexible loop in phosphoribosyltransferases that is essential for purine biosynthesis in cells. These data also provide the framework for designing novel adenine derivatives that could modulate, through hAPRT, diseases-involved cellular pathways.

Coq6 is responsible for the C4-deamination reaction in coenzyme Q biosynthesis in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is able to use para-aminobenzoic acid (pABA) in addition to 4-hydroxybenzoic acid as a precursor of coenzyme Q, a redox lipid essential to the function of the mitochondrial respiratory chain. The biosynthesis of coenzyme Q from pABA requires a deamination reaction at position C4 of the benzene ring to substitute the amino group with an hydroxyl group. We show here that the FAD-dependent monooxygenase Coq6, which is known to hydroxylate position C5, also deaminates position C4 in a reaction implicating molecular oxygen, as demonstrated with labeling experiments. We identify mutations in Coq6 that abrogate the C4-deamination activity, whereas preserving the C5-hydroxylation activity. Several results support that the deletion of Coq9 impacts Coq6, thus explaining the C4-deamination defect observed in Δcoq9 cells. The vast majority of flavin monooxygenases catalyze hydroxylation reactions on a single position of their substrate. Coq6 is thus a rare example of a flavin monooxygenase that is able to act on two different carbon atoms of its C4-aminated substrate, allowing its deamination and ultimately its conversion into coenzyme Q by the other proteins constituting the coenzyme Q biosynthetic pathway.

Overexpression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.

Most of the Coq proteins involved in coenzyme Q (ubiquinone or Q) biosynthesis are interdependent within a multiprotein complex in the yeast Saccharomyces cerevisiae. Lack of only one Coq polypeptide, as in Δcoq strains, results in the degradation of several Coq proteins. Consequently, Δcoq strains accumulate the same early intermediate of the Q(6) biosynthetic pathway; this intermediate is therefore not informative about the deficient biosynthetic step in a particular Δcoq strain. In this work, we report that the overexpression of the protein Coq8 in Δcoq strains restores steady state levels of the unstable Coq proteins. Coq8 has been proposed to be a kinase, and we provide evidence that the kinase activity is essential for the stabilizing effect of Coq8 in the Δcoq strains. This stabilization results in the accumulation of several novel Q(6) biosynthetic intermediates. These Q intermediates identify chemical steps impaired in cells lacking Coq4 and Coq9 polypeptides, for which no function has been established to date. Several of the new intermediates contain a C4-amine and provide information on the deamination reaction that takes place when para-aminobenzoic acid is used as a ring precursor of Q(6). Finally, we used synthetic analogues of 4-hydroxybenzoic acid to bypass deficient biosynthetic steps, and we show here that 2,4-dihydroxybenzoic acid is able to restore Q(6) biosynthesis and respiratory growth in a Δcoq7 strain overexpressing Coq8. The overexpression of Coq8 and the use of 4-hydroxybenzoic acid analogues represent innovative tools to elucidate the Q biosynthetic pathway.

Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.

Coenzyme Q (Q), an essential component of eukaryotic cells, is synthesized by several enzymes from the precursor 4-hydroxybenzoic acid. Mutations in six of the Q biosynthesis genes cause diseases that can sometimes be ameliorated by oral Q supplementation. We establish here that Coq6, a predicted flavin-dependent monooxygenase, is involved exclusively in the C5-hydroxylation reaction. In an unusual way, the ferredoxin Yah1 and the ferredoxin reductase Arh1 may be the in vivo source of electrons for Coq6. We also show that hydroxylated analogs of 4-hydroxybenzoic acid, such as vanillic acid or 3,4-dihydroxybenzoic acid, restore Q biosynthesis and respiration in a Saccharomyces cerevisiae coq6 mutant. Our results demonstrate that appropriate analogs of 4-hydroxybenzoic acid can bypass a deficient Q biosynthetic enzyme and might be considered for the treatment of some primary Q deficiencies.

Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis.

Yeast ubiquinone or coenzyme Q(6) (Q(6)) is a redox active lipid that plays a crucial role in the mitochondrial electron transport chain. At least nine proteins (Coq1p-9p) participate in Q(6) biosynthesis from 4-hydroxybenzoate (4-HB). We now show that the mitochondrial ferredoxin Yah1p and the ferredoxin reductase Arh1p are required for Q(6) biosynthesis, probably for the first hydroxylation of the pathway. Conditional Gal-YAH1 and Gal-ARH1 mutants accumulate 3-hexaprenyl-4-hydroxyphenol and 3-hexaprenyl-4-aminophenol. Para-aminobenzoic acid (pABA) is shown to be the precursor of 3-hexaprenyl-4-aminophenol and to compete with 4-HB for the prenylation reaction catalyzed by Coq2p. Yeast cells convert U-((13)C)-pABA into (13)C ring-labeled Q(6), a result that identifies pABA as a new precursor of Q(6) and implies an additional NH(2)-to-OH conversion in Q(6) biosynthesis. Our study identifies pABA, Yah1p, and Arh1p as three actors in Q(6) biosynthesis.