Structural insights into unique substrate selectivity of Thermoplasma acidophilum D-aldohexose dehydrogenase

The d-aldohexose dehydrogenase from the thermoacidophilic archaea Thermoplasma acidophilum (AldT) belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and catalyzes the oxidation of several monosaccharides with a preference for NAD⁺ rather than NADP⁺ as a cofactor. It has been found that AldT is a unique enzyme that exhibits the highest dehydrogenase activity against d-mannose. Here, we describe the crystal structures of AldT in ligand-free form, in complex with NADH, and in complex with the substrate d-mannose, at 2.1 Å, 1.65 Å, and 1.6 Å resolution, respectively. The AldT subunit forms a typical SDR fold with an unexpectedly long C-terminal tail and assembles into an intertwined tetramer. The d-mannose complex structure reveals that Glu84 interacts with the axial C2 hydroxyl group of the bound d-mannose. Structural comparison with Bacillus megaterium glucose dehydrogenase (BmGlcDH) suggests that the conformation of the glutamate side-chain is crucial for discrimination between d-mannose and its C2 epimer d-glucose, and the conformation of the glutamate side-chain depends on the spatial arrangement of nearby hydrophobic residues that do not directly interact with the substrate. Elucidation of the d-mannose recognition mechanism of AldT further provides structural insights into the unique substrate selectivity of AldT. Finally, we show that the extended C-terminal tail completely shuts the substrate-binding pocket of the neighboring subunit both in the presence and absence of substrate. The elaborate inter-subunit interactions between the C-terminal tail and the entrance of the substrate-binding pocket imply that the tail may play a pivotal role in the enzyme activity.     

Tetratricopeptide repeat proteins Tom70 and Tom71 mediate yeast mitochondrial morphogenesis

The maintenance of correct mitochondrial shape requires numerous proteins that act on the surface or inside of the organelle. Although the soluble F-box protein Mfb1 was recently found to associate peripherally with mitochondria and to regulate organelle connectivity in budding yeast, how it localizes to mitochondria is unknown. Here, we show that two tetratricopeptide repeat proteins-the general preprotein import receptor Tom70 (a component of translocase of the outer membrane) and its paralogue Tom71-are required for Mfb1 mitochondrial localization. Mitochondria in cells lacking Tom70 and Tom71 form short tubules and aggregates, aberrant morphologies similar to those observed in the mfb1-null mutant. In addition, Mfb1 interacts with Tom71 in vivo, and binds to mitochondria through Tom70 in vitro. Our data indicate an unexpected role for Tom70 in recruitment of soluble proteins to the mitochondrial surface, and indicate that Tom71 has a specialized role in Mfb1-mediated mitochondrial morphogenesis.     

Role of RecJ-like protein with 5'-3' exonuclease activity in oligo(deoxy)nucleotide degradation

RecJ-like proteins belonging to the DHH family have been proposed to function as oligoribonucleases and 3'-phosphoadenosine 5'-phosphate (pAp) phosphatases in bacteria and archaea, which do not have Orn (oligoribonuclease) and CysQ (pAp phosphatase) homologs. In this study, we analyzed the biochemical and physiological characterization of the RecJ-like protein TTHA0118 from Thermus thermophilus HB8. TTHA0118 had high enzymatic activity as an oligodeoxyribonucleotide- and oligoribonucleotide-specific exonuclease and as pAp phosphatase. The polarity of degradation was 5' to 3', in contrast to previous reports about Bacillus subtilis NrnA, a RecJ-like protein. TTHA0118 preferentially hydrolyzed short oligodeoxyribonucleotides and oligoribonucleotides, whereas the RecJ exonuclease from T. thermophilus HB8 showed no such length dependence on oligodeoxyribonucleotide substrates. An insertion mutation of the ttha0118 gene led to growth reduction in minimum essential medium. Added 5'-mononucleotides, nucleosides, and cysteine increased growth of the ttha0118 mutant in minimum essential medium. The RecJ-like protein Mpn140 from Mycoplasma pneumoniae M129, which cannot synthesize nucleic acid precursors de novo, showed similar biochemical features to TTHA0118. Furthermore, B. subtilis NrnA also hydrolyzed oligo(deoxy)ribonucleotides in a 5'-3' direction. These results suggested that these RecJ-like proteins act in recycling short oligonucleotides to mononucleotides and in controlling pAp concentrations in vivo.     

Metabolic Quotient Measured by Free-Water Method in Six Enclosures with Different Silver Carp Densities

Quasi-continuous DO and pH measurements (total 47 days) were conducted during enclosure experiments (6 enclosures; 5 × 5 × 2.5 m), in which a biomass gradient of silver carp was created. After subtracting the air–water exchanges of O₂ and CO₂, the chemical and biochemical changes in DO (dissolved oxygen) and DIC (dissolved inorganic carbon) were estimated in order to evaluate MQ (metabolic quotient: DO change divided by DIC change) at intervals of 1 hour. By removing small absolute changes below the threshold value (0.01 mM h^?1), the averaged values of the 24 MQ means for the respective 1-hour periods ranged from 0.96 to 1.20 in the six enclosures. Because the MQs in the daytime inversely correlated well with the ratio of NH⁺ ₄–N to (NH⁺ ₄–N + NO^? ₃–N), not the ecosystems, i.e., density of fish, community structure of zooplankton and phytoplankton, but the form of nitrogen uptaken for primary production principally determined the MQs. The higher MQs observed in the daytime compared with the nighttime (from 14% to 21% except 3% for one enclosure) could not be explained by the denitrification and/or dissolution of CaCO₃ in the sediments, therefore suggesting the selectively faster decomposition of part of the organic matter provided through primary production, in other words, an accumulation of another part of the organic matter in the diurnal and/or daily time scale.     

Solution structure of the RWD domain of the mouse GCN2 protein

GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.     

Genes involved in aniline degradation by Delftia acidovorans strain 7N and its distribution in the natural environment

Aniline-degraders were isolated from activated sludge and environmental samples and classified into eight phylogenetic groups. Seven groups were classified into Gram-negative bacteria, such as Acidovorax sp., Acinetobacter sp., Delftia sp., Comamonas sp., and Pseudomonas sp., suggesting the possible dominance of Gram-negative aniline-degraders in the environment. Aniline degradative genes were cloned from D. acidovorans strain 7N, and the nucleotide sequence of the 8,039-bp fragment containing eight open reading frames was determined. Their deduced amino acid sequences showed homologies to glutamine synthetase (GS)-like protein, glutamine amidotransferase (GA)-like protein, large and small subunits of aniline dioxygenase, reductase, LysR-type regulator, small ferredoxin-like protein, and catechol 2,3-dioxygenase, suggesting a high similarity of this gene cluster to those in P. putida strain UCC22 and Acinetobacter sp. strain YAA. Polymerase chain reaction (PCR) and sequencing analyses of GS-like protein gene segments of other Gram-negative bacteria suggested that Gram-negative bacteria have aniline degradative gene that can be divided into two distinctive groups.     

Serum concentrations of androstenediol and androstenediol sulfate, and their relation to cytokine production during and after normal pregnancy

Since it is known that androstenediol (ADIOL) has potent immunoregulatory effects, changes in ADIOL levels during and after pregnancy might affect the maternal immune system. We examined serum concentrations of ADIOL and androstenediol 3-sulfate (ADIOLS) together with IFN-gamma and IL-4 production levels during pregnancy and after delivery up to 10-11 months postpartum. The subjects were 73 normal pregnant, 76 normal postpartum, and 28 normal non-pregnant women. ADIOL and ADIOLS were measured using EIA and GC/MS, respectively. The cytokine levels in the supernatant of whole-blood cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin were measured using ELISA. ADIOL levels significantly decreased compared to non-pregnant levels in the first trimester (P < 0.05) and were reversed in the third trimester (P < 0.05). After pregnancy, ADIOL levels gradually declined, and a significant decrease was observed at 10-11 months postpartum (P < 0.05). ADIOLS levels were significantly lower in the third trimester (P < 0.05) and significantly higher at the first month postpartum (P < 0.001) compared to non-pregnant women. IFN-gamma and IL-4 levels decreased during pregnancy and subsequently increased postpartum. On the other hand, we found significant negative correlations between ADIOL concentrations and production levels of IFN-gamma (P < 0.05) or IL-4 (P < 0.05). These findings suggest that ADIOL may be involved in modifying the maternal immune response during and after pregnancy.