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Glucuronoxylanases are endo-xylanases and members of the glycoside hydrolase family 30 subfamilies 7 (GH30-7) and 8 (GH30-8). Unlike for the well-studied GH30-8 enzymes, the structural and functional characteristics of GH30-7 enzymes remain poorly understood. Here, we report the catalytic properties and three-dimensional structure of GH30-7 xylanase B (Xyn30B) identified from the cellulolytic fungus Talaromyces cellulolyticus. Xyn30B efficiently degraded glucuronoxylan to acidic xylooligosaccharides (XOSs), including an ␣-1,2-linked 4-O-methyl-D-glucuronosyl substituent (MeGlcA). Rapid analysis with negative-mode electrospray-ionization multistage MS (ESI(؊)-MS n ) revealed that the structures of the acidic XOS products are the same as those of the hydrolysates (MeGlcA 2 Xyl n , n > 2) obtained with typical glucuronoxylanases. Acidic XOS products were further degraded by Xyn30B, releasing first xylobiose and then xylotetraose and xylohexaose as transglycosylation products. This hydrolase reaction was unique to Xyn30B, and the substrate was cleaved at the xylobiose unit from its nonreducing end, indicating that Xyn30B is a bifunctional enzyme possessing both endo-glucuronoxylanase and exo-xylobiohydrolase activities. The crystal structure of Xyn30B was determined as the first structure of a GH30-7 xylanase at 2.25 Å resolution, revealing that Xyn30B is composed of a pseudo-(␣/␤) 8catalytic domain, lacking an ␣6 helix, and a small ␤-rich domain. This structure and site-directed mutagenesis clarified that Arg 46 , conserved in GH30-7 glucuronoxylanases, is a critical residue for MeGlcA appendage-dependent xylan degradation. The structural comparison between Xyn30B and the GH30-8 enzymes suggests that Asn 93 in the ␤2-␣2 loop is involved in xylobiohydrolase activity. In summary, our findings indicate that Xyn30B is a bifunctional endo-and exo-xylanase.The authors declare that they have no conflicts of interest with the contents of this article. This article contains Table S1 and Figs. S1-S6. The atomic coordinates and structure factors (code 6IUJ) have been deposited in the Protein Data Bank (http://wwpdb.org/).

Tryptophan biosynthesis is subject to exquisite control in species of Bacillus and has become one of the best-studied model systems in gene regulation. The protein TRAP (trp RNA-binding attenuation protein) predominantly forms a ring-shaped 11-mer, which binds cognate RNA in the presence of tryptophan to suppress expression of the trp operon. TRAP is itself regulated by the protein Anti-TRAP, which binds to TRAP and prevents RNA binding. To date, the nature of this interaction has proved elusive. Here, we describe mass spectrometry and analytical centrifugation studies of the complex, and 2 crystal structures of the TRAP-Anti-TRAP complex. These crystal structures, both refined to 3.2-Å resolution, show that Anti-TRAP binds to TRAP as a trimer, sterically blocking RNA binding. Mass spectrometry shows that 11-mer TRAP may bind up to 5 AT trimers, and an artificial 12-mer TRAP may bind 6. Both forms of TRAP make the same interactions with Anti-TRAP. Crystallization of wild-type TRAP with Anti-TRAP selectively pulls the 12-mer TRAP form out of solution, so the crystal structure of wild-type TRAP-Anti-TRAP complex reflects a minor species from a mixed population.protein complex ͉ X-ray crystallography ͉ transcription regulation ͉ analytical ultracentrifugation ͉ mass spectrometry T RAP (trp RNA-binding attenuation protein) plays a central role in the highly intricate regulation of transcription and translation of the trp operon in several species of Bacillus, and this system has provided important insights into several mechanisms of gene regulation (1-5). The protein forms an 11-mer ring that binds 11 molecules of tryptophan (Trp) at symmetryrelated sites (6-8), and the TRAP-Trp-RNA complex has been solved for the Bacillus stearothermophilus protein (9). With tryptophan bound, TRAP can bind trp mRNA and induce transcription termination. In Bacillus subtilis, when levels of charged tRNA Trp fall, the protein Anti-TRAP (AT) is expressed, which blocks RNA binding to Trp-bound TRAP and relieves expression of the trp operon (10, 11). AT forms a stable trimer, 4 copies of which can further associate into a dodecamer form (12, 13). Each 53-residue polypeptide chain carries a zincbinding site made of 4 cysteine residues, and chemical crosslinking studies suggested that the active form of AT may be a hexamer (14). Analytical ultracentrifugation data have been interpreted to indicate that the AT dodecamer is the active (TRAP-binding) form, giving an AT 12 -TRAP 11 complex, although it was noted that the data did not rule out alternative stoichiometry (12). Antson and colleagues (13) further proposed, on the basis of their X-ray structure of AT, a model in which the AT dodecamer may bind up to 4 TRAP 11-mer rings by aligning their 11-fold symmetry axes with its 3-fold axes. In this model, AT would not directly block RNA binding, or contact TRAP residues Lys-37 and Arg-58 that are known to be required for TRAP-RNA (15) and TRAP-AT interaction (10). Instead it penetrates the TRAP ring, and it is suggested that there is indirect ...

Insulin-like growth factor-I (IGF-I

The voltage-dependent anion channel (VDAC) is a pore-forming protein expressed in the outer membrane of eukaryotic mitochondria. Three isoforms of it, i.e., VDAC1, VDAC2, and VDAC3, are known to be expressed in mammals; however, the question as to which is the main isoform in mitochondria is still unanswered. To address this question, we first prepared standard VDACs by using a bacterial expression system and raised various antibodies against them by using synthetic peptides as immunogens. Of the three bacterially expressed VDAC isoforms, VDAC3 showed faster migration in SDS-polyacrylamide gels than VDAC1 and VDAC2, although VDAC2 is longer than VDAC1 and VDAC3, due to a 12-amino acid extension of its N-terminal region. Even with careful structural characterization of the expressed VDACs by LC-MS/MS analysis, serious structural modifications of VDACs causing changes in their migration in SDS-polyacrylamide gels were not detected. Next, immunoreactivities of the raised antibodies toward these bacterially expressed VDAC isoforms were evaluated. Trials to prepare specific antibodies against the three individual VDAC isoforms were not successful except in the case of VDAC1. However, using a synthetic peptide corresponding to the highly conserved region among the three VDACs, we were successful in preparing an antibody showing essentially equal immunoreactivities toward all three VDACs. When mitochondrial outer membrane proteins of various rat tissues were subjected to 2-dimensional electrophoresis followed by immunoblotting with this antibody, six immunoreactive protein spots were detected. These spots were characterized by LC-MS/MS analysis, and the signal intensities among the spots were compared. As a result, the signal intensity of the spot representing VDAC1 was the highest, and thus, VDAC1 was concluded to be the most abundantly expressed of the three VDAC isoforms in mammalian mitochondria.

Patients hospitalized in a hospital with a high incidence of antibiotic-associated diarrhea due to toxin A-negative, toxin B-positive (A-/B+) Clostridium difficile were retrospectively investigated to determine the clinical manifestations and risk factors for infection. Of 77 Clostridium difficile isolates obtained from 77 patients during the 1-year investigation period, 30 were A-/B+ and 47 were toxin A-positive, toxin B-positive (A+/B+). By pulsed-field gel electrophoresis analysis, 23 of the 30 A-/B+ strains were outbreak-related, suggesting nosocomial spread of a single type of bacterium, which mainly affected patients in the wards of respiratory medicine, hematology and neurology. Using regression analysis, three factors were found to be associated with infection by A-/B+ isolates: (i) exposure to antineoplastic agents ( P=0.01, odds ratio [OR]=5.1), (ii) the use of nasal feeding tubes ( P=0.008, OR=5.2), and (iii) assignment to a certain internal medicine ward ( P=0.05, OR=3.0). Between patients with Clostridium difficile-associated diarrhea caused by A-/B+ strains and those with A+/B+ strains, no statistically significant difference was found in body temperature, serum concentration of C-reactive protein, leukocyte count in whole blood, frequency of diarrhea, or type of underlying disease. These results indicate that A-/B+ strains of Clostridium difficile can cause intestinal infection in humans and they spread nosocomially in the same manner as A+/B+ strains.

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