Precise control of mitochondrial DNA gene expression is critical for regulation of oxidative phosphorylation capacity in mammals. The MTERF protein family plays a key role in this process, and its members have been implicated in regulation of transcription initiation and site-specific transcription termination. We now demonstrate that a member of this family, MTERF4, directly controls mitochondrial ribosomal biogenesis and translation. MTERF4 forms a stoichiometric complex with the ribosomal RNA methyltransferase NSUN4 and is necessary for recruitment of this factor to the large ribosomal subunit. Loss of MTERF4 leads to defective ribosomal assembly and a drastic reduction in translation. Our results thus show that MTERF4 is an important regulator of translation in mammalian mitochondria.
Pyranose 2-oxidase (P2Ox) participates in fungal lignin degradation by producing the H 2 O 2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose-and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to the corresponding ketoaldoses. To investigate the structural determinants of catalysis, covalent flavinylation, substrate binding, and regioselectivity, wild-type and mutant P2Ox enzymes were produced and characterized biochemically and structurally. Removal of the histidyl-FAD linkage resulted in a catalytically competent enzyme containing tightly, but noncovalently bound FAD. This mutant (H167A) is characterized by a 5-fold lower k cat , and a 35-mV lower redox potential, although no significant structural changes were seen in its crystal structure. In previous structures of P2Ox, the substrate loop (residues 452-457) covering the active site has been either disordered or in a conformation incompatible with carbohydrate binding. We present here the crystal structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-D-glucose. Pyranose 2-oxidase (P2Ox, 3 pyranose:oxygen 2-oxidoreductase; glucose 2-oxidase; EC 1.1.3.10) is a flavin adenine dinucleotide (FAD)-dependent oxidase present in the hyphal periplasmic space (1) of wood-degrading basidiomycetes (2, 3). These fungi are the only known microorganisms that are capable of fully mineralizing lignin, and P2Ox has a proposed role in the oxidative events (4) of lignin degradation by providing the essential co-substrate, H 2 O 2 , for lignin and manganese peroxidases (5, 6). An alternative hypothesis assigns a role for P2Ox in both H 2 O 2 production and in the reduction of quinones in the periplasm or in the extracellular environment (7). P2Ox from the white-rot fungi Trametes multicolor (Trametes ochracea) and Peniophora gigantea are hitherto the most studied biochemically (7-10) and structurally (11, 12).P2Ox oxidizes a broad range of carbohydrate substrates that are natural constituents of hemicelluloses, allowing most lignocellulose-derived sugars to be utilized. Substrates can be oxidized regioselectively at the C2 position, although some oxidation at C3 can occur as a side reaction (10). For C2 oxidation, D-glucose, D-xylose, and L-sorbose are good or reasonably good substrates, and D-galactose and L-arabinose perform poorly as substrates (7). Based on the catalytic efficiency, k cat /K m , D-glucose (D-Glc) is the best substrate for T. multicolor P2Ox (7). Substrates that are oxidized at C3 were analyzed for P. gigantea P2Ox and include 2-deoxy-D-glucose, 2-keto-D-glucose, and methyl -D-glucosides (13, 10). That oxidation can take place either at C2 or at C3 presupposes two distinct, productive binding modes (referred to here as C2 ox and C3 ox ) for a monosaccharide in the P2Ox active site.P2Ox from T. multicolor is homotetrameric with a molecular mass of 270 kDa (7) where each of the four subunits carries one FAD molecule bound covalently to N ⑀2 (i.e. N3) of His 167 via its 8␣-methyl group (14, 11). The...
Presequence protease PreP is a novel protease that degrades targeting peptides as well as other unstructured peptides in both mitochondria and chloroplasts. The first structure of PreP from Arabidopsis thaliana refined at 2.1 Å resolution shows how the 995‐residue polypeptide forms a unique proteolytic chamber of more than 10 000 Å3 in which the active site resides. Although there is no visible opening to the chamber, a peptide is bound to the active site. The closed conformation places previously unidentified residues from the C‐terminal domain at the active site, separated by almost 800 residues in sequence to active site residues located in the N‐terminal domain. Based on the structure, a novel mechanism for proteolysis is proposed involving hinge‐bending motions that cause the protease to open and close in response to substrate binding. In support of this model, cysteine double mutants designed to keep the chamber covalently locked show no activity under oxidizing conditions. The manner in which substrates are processed inside the chamber is reminiscent of the proteasome; therefore, we refer to this protein as a peptidasome.
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