Nitric oxide reductase (NOR) is an iron-containing enzyme that catalyzes the reduction of nitric oxide (NO) to generate a major greenhouse gas, nitrous oxide (N(2)O). Here, we report the crystal structure of NOR from Pseudomonas aeruginosa at 2.7 angstrom resolution. The structure reveals details of the catalytic binuclear center. The non-heme iron (Fe(B)) is coordinated by three His and one Glu ligands, but a His-Tyr covalent linkage common in cytochrome oxidases (COX) is absent. This structural characteristic is crucial for NOR reaction. Although the overall structure of NOR is closely related to COX, neither the D- nor K-proton pathway, which connect the COX active center to the intracellular space, was observed. Protons required for the NOR reaction are probably provided from the extracellular side.

Recent articles have reported an association between fatty liver disease and systemic insulin resistance in humans, but the causal relationship remains unclear. The liver may contribute to muscle insulin resistance by releasing secretory proteins called hepatokines. Here we demonstrate that leukocyte cell–derived chemotaxin 2 (LECT2), an energy-sensing hepatokine, is a link between obesity and skeletal muscle insulin resistance. Circulating LECT2 positively correlated with the severity of both obesity and insulin resistance in humans. LECT2 expression was negatively regulated by starvation-sensing kinase adenosine monophosphate-activated protein kinase in H4IIEC hepatocytes. Genetic deletion of LECT2 in mice increased insulin sensitivity in the skeletal muscle. Treatment with recombinant LECT2 protein impaired insulin signaling via phosphorylation of Jun NH2-terminal kinase in C2C12 myocytes. These results demonstrate the involvement of LECT2 in glucose metabolism and suggest that LECT2 may be a therapeutic target for obesity-associated insulin resistance.

The structure of quinol-dependent nitric oxide reductase (qNOR) from G. stearothermophilus, which catalyzes the reduction of NO to produce the major ozone-depleting gas N(2)O, has been characterized at 2.5 Å resolution. The overall fold of qNOR is similar to that of cytochrome c-dependent NOR (cNOR), and some structural features that are characteristic of cNOR, such as the calcium binding site and hydrophilic cytochrome c domain, are observed in qNOR, even though it harbors no heme c. In contrast to cNOR, structure-based mutagenesis and molecular dynamics simulation studies of qNOR suggest that a water channel from the cytoplasm can serve as a proton transfer pathway for the catalytic reaction. Further structural comparison of qNOR with cNOR and aerobic and microaerobic respiratory oxidases elucidates their evolutionary relationship and possible functional conversions.

ER-associated degradation (ERAD) is an ER quality-control process that eliminates terminally misfolded proteins. ERdj5 was recently discovered to be a key ER-resident PDI family member protein that accelerates ERAD by reducing incorrect disulfide bonds in misfolded glycoproteins recognized by EDEM1. We here solved the crystal structure of full-length ERdj5, thereby revealing that ERdj5 contains the N-terminal J domain and six tandem thioredoxin domains that can be divided into the N- and C-terminal clusters. Our systematic biochemical analyses indicated that two thioredoxin domains that constitute the C-terminal cluster form the highly reducing platform that interacts with EDEM1 and reduces EDEM1-recruited substrates, leading to their facilitated degradation. The pulse-chase experiment further provided direct evidence for the sequential movement of an ERAD substrate from calnexin to the downstream EDEM1-ERdj5 complex, and then to the retrotranslocation channel, probably through BiP. We present a detailed molecular view of how ERdj5 mediates ERAD in concert with EDEM1.

It remains unclear how hepatic steatosis links to inflammation. Leukocyte cell-derived chemotaxin 2 (LECT2) is a hepatokine that senses fat in the liver and is upregulated prior to weight gain. The aim of this study was to investigate the significance of LECT2 in the development of nonalcoholic steatohepatitis (NASH). In human liver biopsy samples, elevated LECT2 mRNA levels were positively correlated with body mass index (BMI) and increased in patients who have steatosis and inflammation in the liver. LECT2 mRNA levels were also positively correlated with the mRNA levels of the inflammatory genes CCR2 and TLR4. In C57BL/6J mice fed with a high-fat diet, mRNA levels of the inflammatory cytokines Tnfa and Nos2 were significantly lower in Lect2 KO mice. In flow cytometry analyses, the number of M1-like macrophages and M1/M2 ratio were significantly lower in Lect2 KO mice than in WT mice. In KUP5, mouse kupffer cell line, LECT2 selectively enhanced the LPS-induced phosphorylation of JNK, but not that of ERK and p38. Consistently, LECT2 enhanced the LPS-induced phosphorylation of MKK4 and TAB2, upstream activators of JNK. Hepatic expression of LECT2 is upregulated in association with the inflammatory signature in human liver tissues. The elevation of LECT2 shifts liver residual macrophage to the M1-like phenotype, and contributes to the development of liver inflammation. These findings shed light on the hepatokine LECT2 as a potential therapeutic target that can dissociate liver steatosis from inflammation.

Cytochrome bd is a heterodimeric terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. For understanding the unique catalytic mechanism of the quinol oxidation, mass spectrometry was used to identify amino acid residue(s) that can be labeled with a reduced form of 2-azido-3-methoxy-5-methyl-6-geranyl-1,4-benzoquinone or 2-methoxy-3-azido-5-methyl-6-geranyl-1,4-benzoquinone. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated that the photo inactivation of ubiquinol-1 oxidase activity was accompanied by the labeling of subunit I with both azidoquinols. The cross- Cytochrome bd (CydAB) is a heterodimeric ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and is predominantly expressed under microaerophilic growth conditions (see Refs. 1-3 for reviews). It catalyzes dioxygen reduction with two molecules of ubiquinol-8 (Q 8 H 2 ), 2 leading to the release of four protons from quinols to the periplasm. Through a putative proton channel, four protons used for dioxygen reduction are taken up from the cytoplasm and delivered to the dioxygen reduction site at the periplasmic side of the cytoplasmic membrane (4). During dioxygen reduction, cytochrome bd generates an electrochemical proton gradient (⌬pH and membrane potential) across the membrane through apparent transmembrane movement of four chemical protons (5-7). In contrast to cytochrome bo, an alternative oxidase under highly aerated growth conditions, cytochrome bd has no proton pumping activity and does not belong to the heme-copper terminal oxidase superfamily.On the basis of spectroscopic and ligand binding studies, three distinct redox metal centers have been identified as heme b 558 , heme b 595 , and heme d (see Ref. 8 for a review). Unlike cytochrome bo, cytochrome bd does not contain a tightly bound Q 8 . Heme b 558 is a low spin protoheme IX and is ligated by I-His 186 (helix V) and I-Met 393 (helix VII) of subunit I (CydA) (9). Heme b 595 is a high spin protoheme IX bound to I-His 19 (helix I) of subunit I (9) and mediates electron transfer from heme b 558 to heme d, where dioxygen is reduced to water (10 -13). Heme d is a high spin chlorin bound to an unidentified nitrogenous ligand (14 -16) and forms a di-heme binuclear center with heme b 595 (16,17). Topological analysis suggests that all of the hemes are located at the periplasmic end of transmembrane helices (4).In loop VI/VII (Q-loop) of subunit I, binding of monoclonal antibodies to 252 KLAAIEAEWET 262 (18,19) and proteolytic cleavage with trypsin at I-Tyr 290 or chymotrypsin at I-Arg 298 (20, 21) suppressed ubiquinol oxidase activity. Photoaffinity labeling studies with 2-methyl-3-azido-5-methoxy-6-(3,7-dimethyl-[ 3 H]octyl)-1,4-benzoquinone ([ 3 H]3-azido-2-methyl-5-methoxy-BQ 2s ) indicate the presence of the quinol oxidation site in subunit I (22). These results suggest that periplasmic loop VI/VII in subunit I is involved in the ubiquinol oxidation site. Inhibitor binding studies indicated the close proximity ...

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Chemical investigation of the Okinawan soft coral Clavularia koellikeri resulted in the isolation of two new cembrane diterpenoids (1 and 2) and one new dollabelane diterpenoid, 3. Their structures were determined on the basis of the results of spectroscopic analysis. Compounds 1 and 3 were examined for in vitro growth-inhibition effects toward tumor cells.