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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.

Proteins crucial for the respiratory chain are translated by the mitochondrial ribosome. Mitochondrial ribosome biogenesis is therefore critical for oxidative phosphorylation capacity and disturbances are known to cause human disease. This complex process is evolutionary conserved and involves several RNA processing and modification steps required for correct ribosomal RNA maturation. We recently showed that a member of the mitochondrial transcription termination factor (MTERF) family of proteins, MTERF4, recruits NSUN4, a 5-methylcytosine RNA methyltransferase, to the large ribosomal subunit in a process crucial for mitochondrial ribosome biogenesis. Here, we describe the 3D crystal structure of the human MTERF4-NSUN4 complex determined to 2.9 Å resolution. MTERF4 is composed of structurally repeated MTERF-motifs that form a nucleic acid binding domain. NSUN4 lacks an N-or C-terminal extension that is commonly used for RNA recognition by related RNA methyltransferases. Instead, NSUN4 binds to the C-terminus of MTERF4. A positively charged surface forms an RNA binding path from the concave to the convex side of MTERF4 and further along NSUN4 all of the way into the active site. This finding suggests that both subunits of the protein complex likely contribute to RNA recognition. The interface between MTERF4 and NSUN4 contains evolutionarily conserved polar and hydrophobic amino acids, and mutations that change these residues completely disrupt complex formation. This study provides a molecular explanation for MTERF4-dependent recruitment of NSUN4 to ribosomal RNA and suggests a unique mechanism by which other members of the large MTERF-family of proteins can regulate ribosomal biogenesis. mitochondria | translation | X-ray crystallography

The extracellular flavocytochrome cellobiose dehydrogenase (CDH; EC 1.1.99.18) participates in lignocellulose degradation by white-rot fungi with a proposed role in the early events of wood degradation. The complete hemoflavoenzyme consists of a catalytically active dehydrogenase fragment (DH cdh ) connected to a b-type cytochrome domain via a linker peptide. In the reductive half-reaction, DH cdh catalyzes the oxidation of cellobiose to yield cellobiono-1,5-lactone. The active site of DH cdh is structurally similar to that of glucose oxidase and cholesterol oxidase, with a conserved histidine residue positioned at the re face of the flavin ring close to the N5 atom. The mechanisms of oxidation in glucose oxidase and cholesterol oxidase are still poorly understood, partly because of lack of experimental structure data or difficulties in interpreting existing data for enzyme-ligand complexes. Here we report the crystal structure of the Phanerochaete chrysosporium DH cdh with a bound inhibitor, cellobiono-1,5-lactam, at 1.8-Å resolution. The distance between the lactam C1 and the flavin N5 is only 2.9 Å, implying that in an approximately planar transition state, the maximum distance for the axial 1-hydrogen to travel for covalent addition to N5 is 0.8 -0.9 Å. The lactam O1 interacts intimately with the side chains of His-689 and Asn-732. Our data lend substantial structural support to a reaction mechanism where His-689 acts as a general base by abstracting the O1 hydroxyl proton in concert with transfer of the C1 hydrogen as hydride to the re face of the flavin N5.Cellobiose dehydrogenases (CDHs 1 ; EC 1.1.99.18) are extracellular fungal flavocytochromes that are believed to participate in lignocellulose degradation by fungi. They are oxidoreductases carrying protoheme and FAD cofactors bound to separate domains. In vitro, CDH from the white-rot Basidiomycete Phanerochaete chrysosporium depolymerizes cellulose, hemicelluloses, and lignin (Refs. 1-3; for review, see Ref. 4) as well as other polymers (5). The exact biological function of CDH has been a subject of lively debate, but recent results suggest that the enzyme is important for invasion and colonization of wood (6).The catalytic site is located in the flavoprotein domain, where the reductive half-reaction proceeds by oxidation of ␤-cellobiose (apparent k cat 15.7 s Ϫ1 and K m 0.11 mM, see Ref. 7) to yield cellobiono-1,5-lactone (Fig.

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Altered inositol metabolism is implicated in a number of diabetic complications. The first committed step in mammalian inositol catabolism is performed by myo-inositol oxygenase (MIOX), which catalyzes a unique four-electron dioxygen-dependent ring cleavage of myo-inositol to D-glucuronate. Here, we present the crystal structure of human MIOX in complex with myo-inosose-1 bound in a terminal mode to the MIOX diiron cluster site. Furthermore, from biochemical and biophysical results from N-terminal deletion mutagenesis we show that the N terminus is important, through coordination of a set of loops covering the active site, in shielding the active site during catalysis. EPR spectroscopy of the unliganded enzyme displays a two-component spectrum that we can relate to an open and a closed active site conformation. Furthermore, based on site-directed mutagenesis in combination with biochemical and biophysical data, we propose a novel role for Lys 127 in governing access to the diiron cluster.myo-Inositol is a cyclitol that plays a crucial role in all eukaryotic cells by serving as a backbone for the most important second messengers: inositol phosphates and their lipid derivatives, phosphoinositides. Although inositol phosphates and phosphoinositides have been and continue to be the focus of intense study, the mechanisms for maintenance of total cellular inositol levels and the medical implications of deranged intracellular inositol levels are less studied. It is known that inositol homeostasis is often disturbed in diabetes (1) and that intracellular depletion of myo-inositol is associated with common diabetic complications, such as cataracts, nephropathies, retinopathies, and neuropathies (2). In mammalian cells, the only pathway for inositol breakdown utilizes myo-inositol oxygenase (MIOX) 3 to catalyze the first committed step by a dioxygen-dependent cleavage between C1 and C6 of the inositol ring to form D-glucuronate, which can then enter the glucuronate-xylulose pathway (2). Therapeutic intervention aimed at inhibiting MIOX activity may be a future cure for diabetic complications caused by inositol depletion.18 O labeling studies have shown that MIOX incorporates only a single oxygen into the product and that oxygen is found exclusively in the D-glucuronate carboxylate group. MIOX has a pH optimum of 9.5 (3) with myo-inositol (K m ϭ 5.9 mM; k cat ϭ 11 min Ϫ1 (2)) and its epimer D-chiroinositol (K m ϭ 33 mM; k cat ϭ 2.3 min Ϫ1 (2)) as its only known substrates. Moreover, the only known reasonably potent inhibitor is myo-inosose-1 (K i ϭ 62 M (3)). It has been noted that an N-terminally truncated fragment starting at Thr 32 is formed upon storage of recombinant protein (4). Interestingly, this uncharacterized N-terminal fragment is also observed in vivo (5).It was recently shown, using EPR and Mössbauer spectroscopy, that MIOX is a new member of the nonheme diiron-dependent dioxygen-activating family (6). Interestingly, the sequence indicated strong dissimilarity to other members of this family (6) that all fe...