Browsing by Subject "SYNTHETASE"

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  • Olsen, Rikke K. J.; Konarikova, Eliska; Giancaspero, Teresa A.; Mosegaard, Signe; Boczonadi, Veronika; Matakovic, Lavinija; Veauville-Merllie, Alice; Terrile, Caterina; Schwarzmayr, Thomas; Haack, Tobias B.; Auranen, Mari; Leone, Piero; Galluccio, Michele; Imbard, Apolline; Gutierrez-Rios, Purificacion; Palmfeldt, Johan; Graf, Elisabeth; Vianey-Saban, Christine; Oppenheim, Marcus; Schiff, Manuel; Pichard, Samia; Rigal, Odile; Pyle, Angela; Chinnery, Patrick F.; Konstantopoulou, Vassiliki; Moslinger, Dorothea; Feichtinger, Rene G.; Talim, Beril; Topaloglu, Haluk; Coskun, Turgay; Gucer, Safak; Botta, Annalisa; Pegoraro, Elena; Malena, Adriana; Vergani, Lodovica; Mazza, Daniela; Zollino, Marcella; Ghezzi, Daniele; Acquaviva, Cecile; Tyni, Tiina; Boneh, Avihu; Meitinger, Thomas; Strom, Tim M.; Gregersen, Niels; Mayr, Johannes A.; Horvath, Rita; Barile, Maria; Prokisch, Holger (2016)
    Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis.
  • Zaretsky, Marianna; Roine, Elina; Eichler, Jerry (2018)
    N-glycosylation is a post-translational modification that occurs in all three domains. In Archaea, however, N-linked glycans present a degree of compositional diversity not observed in either Eukarya or Bacteria. As such, it is surprising that nonulosonic acids (NulOs), nine-carbon sugars that include sialic acids, pseudaminic acids, and legionaminic acids, are routinely detected as components of protein-linked glycans in Eukarya and Bacteria but not in Archaea. In the following, we report that the N-linked glycan attached to the S-layer glycoprotein of the haloarchaea Halorubrum sp. PV6 includes an N-formylated legionaminic acid. Analysis of the Halorubrum sp. PV6 genome led to the identification of sequences predicted to comprise the legionaminic acid biosynthesis pathway. The transcription of pathway genes was confirmed, as was the co-transcription of several of these genes. In addition, the activities of LegI, which catalyzes the condensation of 2,4-di-N-acetyl-6-deoxymannose and phosphoenolpyruvate to generate legionaminic acid, and LegF, which catalyzes the addition of cytidine monophosphate (CMP) to legionaminic acid, both heterologously expressed in Haloferax volcanii, were demonstrated. Further genome analysis predicts that the genes encoding enzymes of the legionaminic acid biosynthetic pathway are clustered together with sequences seemingly encoding components of the N-glycosylation pathway in this organism. In defining the first example of a legionaminic acid biosynthesis pathway in Archaea, the findings reported here expand our insight into archaeal N-glycosylation, an almost universal post-translational modification in this domain of life.
  • Savijoki, Kirsi; Myllymäki, Henna; Luukinen, Hanna; Paulamäki, Lauri; Vanha-aho, Leena-Maija; Svorjova, Aleksandra; Miettinen, Ilkka; Fallarero, Adyary; Ihalainen, Teemu O; Yli-Kauhaluoma, Jari; Nyman, Tuula A.; Parikka, Mataleena (2021)
    The complex cell wall and biofilm matrix (ECM) act as key barriers to antibiotics in mycobacteria. Here, the ECM and envelope proteins of Mycobacterium marinum ATCC 927, a nontuberculous mycobacterial model, were monitored over 3 months by label-free proteomics and compared with cell surface proteins on planktonic cells to uncover pathways leading to virulence, tolerance, and persistence. We show that ATCC 927 forms pellicle-type and submerged-type biofilms (PBFs and SBFs, respectively) after 2 weeks and 2 days of growth, respectively, and that the increased CelA1 synthesis in this strain prevents biofilm formation and leads to reduced rifampicin tolerance. The proteomic data suggest that specific changes in mycolic acid synthesis (cord factor), Esx1 secretion, and cell wall adhesins explain the appearance of PBFs as ribbon-like cords and SBFs as lichen-like structures. A subpopulation of cells resisting 64x MIC rifampicin (persisters) was detected in both biofilm subtypes and already in 1-week-old SBFs. The key forces boosting their development could include subtype-dependent changes in asymmetric cell division, cell wall biogenesis, tricarboxylic acid/glyoxylate cycle activities, and energy/redox/iron metabolisms. The effect of various ambient oxygen tensions on each cell type and nonclassical protein secretion are likely factors explaining the majority of the subtype-specific changes. The proteomic findings also imply that Esx1-type protein secretion is more efficient in planktonic (PL) and PBF cells, while SBF may prefer both the Esx5 and nonclassical pathways to control virulence and prolonged viability/persistence. In conclusion, this study reports the first proteomic insight into aging mycobacterial biofilm ECMs and indicates biofilm subtype-dependent mechanisms conferring increased adaptive potential and virulence of nontuberculous mycobacteria. IMPORTANCE Mycobacteria are naturally resilient, and mycobacterial infections are notoriously difficult to treat with antibiotics, with biofilm formation being the main factor complicating the successful treatment of tuberculosis (TB). The present study shows that nontuberculous Mycobacterium marinum ATCC 927 forms submergedand pellicle-type biofilms with lichen- and ribbon-like structures, respectively, as well as persister cells under the same conditions. We show that both biofilm subtypes differ in terms of virulence-, tolerance-, and persistence-conferring activities, highlighting the fact that both subtypes should be targeted to maximize the power of antimycobacterial treatment therapies.