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  • Recently Schenck et al identified an


    Recently, Schenck et al. [25] identified an active site asparagine 222 (N222) residue in the Glycine max TyrAp/PDH (GmPDH1) that is responsible for its prephenate substrate specificity. Mutating the single N222 into an acidic residue (i.e. aspartate, D) in the legume TyrAp/PDH shifted from prephenate to arogenate substrate specificity. The mutation also introduced partial tyrosine sensitivity to otherwise completely insensitive GmPDH1. Introduction of an asparagine (N) residue at the corresponding position in TyrAa/ADH enzymes from non-legume plants [25] and some bacteria [26] also conferred prephenate substrate specificity. Thus, the architecture of the tyrosine biosynthetic pathways can be different between and within taxonomic groups due to the substrate preference of TyrA enzymes, which can be altered by a single mutation in plants and some bacteria. The tyrosine and phenylalanine biosynthetic pathways have been previously studied in the fungi Candida maltosa [41], Saccharomyces cerevisiae [42], Neurospora sp. [35], and Claviceps paspali [43]. These studies detected only prephenate dehydrogenase activity from the protein extracts of these fungal species. The prephenate dehydrogenase activities from crude extracts of C. maltosa and Neurospora sp. were inhibited by tyrosine but not from those of S. cerevisiae and C. paspali [[41], [42], [43]]. Although a TyrA gene has been reported from S. cerevisiae (as TYR1) [44], its gene product has not been biochemically characterized. Hence, it is not fully understood how tyrosine is synthesized in fungi. Here, we characterized recombinant TyrA enzymes from species in two major fungal phyla, Ascomycota and Basidiomycota. The yeast Saccharomyces cerevisiae is a model organism of eukaryotes [45,46] while the filamentous fungus Pleurotus ostreatus is economically important, not only as an edible mushroom but also because of its ability to degrade lignin [[47], [48], [49]]. We found that tyrosine is produced by the prephenate-specific TyrA enzymes in both organisms; however, their TyrA enzymes showed distinct feedback regulation by tyrosine. Site-directed mutagenesis of the previously-reported residue that confers substrate specificity of plant TyrA enzymes [25,26] did not alter the substrate specificity of ScTyrA, suggesting that as yet unknown amino zilpaterol residue(s) is responsible for the substrate specificity of fugal TyrA enzymes.
    Materials and methods
    Results and discussion
    Conflicts of interest statement
    Acknowledgements We thank Bastien Bennetot for his assistance in recombinant protein isolation. This work was supported by the National Science Foundation grant IOS-1354971 and an Agriculture and Food Research Initiative competitive grant (2015-67013-22955) from the USDA National Institute of Food and Agriculture to H.A.M. S.L.N. was partly supported by the National Science Foundation Graduate Research Fellowship (Grant No. DGE-1256259) and an Advanced Opportunity Fellowship (AOF) from the University of Wisconsin-Madison.
    Introduction Moonlighting of metabolic enzymes in the nucleus has been recognized as an important mechanism linking metabolic flux to regulation of gene expression. Dynamic nuclear translocation of pyruvate dehydrogenase complex (PDHC) and increased acetyl-CoA affect histone acetylation and epigenetic regulation. Lactate dehydrogenase (LDH) is also detected in the nucleus[3], [4] where it generates lactate. Lactate has global effects on gene transcription which overlap with the changes induced by histone deacetylase (HDAC) inhibitors.
    Materials and methods
    Discussion Translocations of metabolic enzymes to the nucleus link metabolism to gene expression. In this study, we found nuclear translocations of PDHC and LDH in mice with liver injury induced by CD95-Ab, α-amanitin, and APAP. Hepatocytes are very sensitive to Fas-induced apoptosis and CD95-Ab results in rapid death in mice due to fulminant hepatitis, mimicking acute liver failure in humans. The increased levels of nPDHC and nLDH in livers were associated with higher levels of their products acetyl-CoA and lactate in nuclear fractions, marked increase in histone acetylation and changes in gene expression. Acetyl-CoA is a membrane impermeable and unstable metabolite required for histone acetylation. Increased nuclear lactate is known to have global effects on gene expression via inhibition of HDAC. Because changes in expression affected genes related to damage response with negative consequences on cell survival, we sought to inhibit nLDH and nPDHC activity in mice with acute liver failure. The reduced liver damage and increased survival of mice treated with the histone acetyltransferase inhibitor garcinol support the detrimental role of histone hyper-acetylation during acute liver failure.