The α- and ß-Subunit Boundary at the Stem of the Mushroom-Like α3ß3-Type Oxygenase Component of Rieske Non-Heme Iron Oxygenases Is the Rieske-Type Ferredoxin-Binding Site.

Cumene dioxygenase (CumDO) is an initial enzyme in the cumene degradation pathway of Pseudomonas fluorescens IP01 and is a Rieske non-heme iron oxygenase (RO) that comprises two electron transfer components (reductase [CumDO-R] and Rieske-type ferredoxin [CumDO-F]) and one catalytic component (α3ß3-type oxygenase [CumDO-O]). Catalysis is triggered by electrons that are transferred from NAD(P)H to CumDO-O by CumDO-R and CumDO-F. To investigate the binding mode between CumDO-F and CumDO-O and to identify the key CumDO-O amino acid residues for binding, we simulated docking between the CumDO-O crystal structure and predicted model of CumDO-F and identified two potential binding sites: one is at the side-wise site and the other is at the top-wise site in mushroom-like CumDO-O. Then, we performed alanine mutagenesis of 16 surface amino acid residues at two potential binding sites. The results of reduction efficiency analyses using the purified components indicated that CumDO-F bound at the side-wise site of CumDO-O, and K117 of the α-subunit and R65 of the ß-subunit were critical for the interaction. Moreover, these two positively charged residues are well conserved in α3ß3-type oxygenase components of ROs whose electron donors are Rieske-type ferredoxins. Given that these residues were not conserved if the electron donors were different types of ferredoxins or reductases, the side-wise site of the mushroom-like structure is thought to be the common binding site between Rieske-type ferredoxin and α3ß3-type oxygenase components in ROs. IMPORTANCE We clarified the critical amino acid residues of the oxygenase component (Oxy) of Rieske non-heme iron oxygenase (RO) for binding with Rieske-type ferredoxin (Fd). Our results showed that Rieske-type Fd-binding site is commonly located at the stem (side-wise site) of the mushroom-like α3ß3 quaternary structure in many ROs. The resultant binding site was totally different from those reported at the top-wise site of the doughnut-like α3-type Oxy, although α3-type Oxys correspond to the cap (α3 subunit part) of the mushroom-like α3ß3-type Oxys. Critical amino acid residues detected in this study were not conserved if the electron donors of Oxys were different types of Fds or reductases. Altogether, we can suggest that unique binding modes between Oxys and electron donors have evolved, depending on the nature of the electron donors, despite Oxy molecules having shared α3ß3 quaternary structures.

Increase in muscle mass associated with the prebiotic effects of 1-kestose in super-elderly patients with sarcopenia.

Sarcopenia causes functional disorders and decreases the quality of life. Thus, it has attracted substantial attention in the aging modern world. Dysbiosis of the intestinal microbiota is associated with sarcopenia; however, it remains unclear whether prebiotics change the microbiota composition and result in the subsequent recovery of muscle atrophy in elderly patients with sarcopenia. This study aimed to assess the effects of prebiotics in super-elderly patients with sarcopenia. We analyzed the effects of 1-kestose on the changes in the intestinal microbiota and body composition using a next-generation sequencer and a multi-frequency bioimpedance analysis device. The Bifidobacterium longum population was significantly increased in the intestine after 1-kestose administration. In addition, in all six patients after 12 weeks of 1-kestose administration, the skeletal muscle mass index was greater, and the body fat percentage was lower. This is the first study to show that administration of a prebiotic increased the population of B. longum in the intestinal microbiota and caused recovery of muscle atrophy in super-elderly patients with sarcopenia.

Structural similarities and differences in H-NS family proteins revealed by the N-terminal structure of TurB in Pseudomonas putida KT2440.

H-NS family proteins play key roles in bacterial nucleoid compaction and global transcription. MvaT homologues in Pseudomonas have almost negligible amino acid sequence identity with H-NS, but can complement an hns-related phenotype of Escherichia coli. Here, we report the crystal structure of the N-terminal dimerization/oligomerization domain of TurB, an MvaT homologue in Pseudomonas putida KT2440. Our data identify two dimerization sites; the structure of the central dimerization site is almost the same as the corresponding region of H-NS, whereas the terminal dimerization sites are different. Our results reveal similarities and differences in dimerization and oligomerization mechanisms between H-NS and TurB.

Purification and partial characterization of the extradiol dioxygenase, 2'-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase, in the fluorene degradation pathway from Rhodococcus sp. strain DFA3.

Type II extradiol dioxygenase, 2'-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase (FlnD1D2) involved in the fluorene degradation pathway of Rhodococcus sp. DFA3 was purified to homogeneity from a heterologously expressing Escherichia coli. Gel filtration chromatography and SDS-PAGE suggested that FlnD1D2 is an α4ß4 heterooctamer and that the molecular masses of these subunits are 30 and 9.9 kDa, respectively. The optimum pH and temperature for enzyme activity were 8.0 and 30 °C, respectively. Assessment of metal ion effects suggested that exogenously supplied Fe(2+) increases enzyme activity 3.2-fold. FlnD1D2 catalyzed meta-cleavage of 2'-carboxy-2,3-dihydroxybiphenyl homologous compounds, but not single-ring catecholic compounds. The Km and kcat/Km values of FlnD1D2 for 2,3-dihidroxybiphenyl were 97.2 µM and 1.5 × 10(-2) µM(-1)sec(-1), and for 2,2',3-trihydroxybiphenyl, they were 168.0 µM and 0.5 × 10(-2) µM(-1)sec(-1), respectively. A phylogenetic tree of the large and small subunits of type II extradiol dioxygenases suggested that FlnD1D2 constitutes a novel subgroup among heterooligomeric type II extradiol dioxygenases.

Crystallization and preliminary X-ray diffraction analyses of the redox-controlled complex of terminal oxygenase and ferredoxin components in the Rieske nonhaem iron oxygenase carbazole 1,9a-dioxygenase.

The initial reaction in bacterial carbazole degradation is catalyzed by carbazole 1,9a-dioxygenase, which consists of terminal oxygenase (Oxy), ferredoxin (Fd) and ferredoxin reductase components. The electron-transfer complex between reduced Oxy and oxidized Fd was crystallized at 293 K using the hanging-drop vapour-diffusion method with PEG 3350 as the precipitant under anaerobic conditions. The crystal diffracted to a maximum resolution of 2.25 Šand belonged to space group P21, with unit-cell parameters a = 97.3, b = 81.6, c = 116.2 Å, α = γ = 90, ß = 100.1°. The VM value is 2.85 Å(3) Da(-1), indicating a solvent content of 56.8%.

Crystallization and preliminary X-ray diffraction studies of the reduced form of the terminal oxygenase component of the Rieske nonhaem iron oxygenase system carbazole 1,9a-dioxygenase.

The initial reaction of bacterial carbazole degradation is catalysed by carbazole 1,9a-dioxygenase, which consists of terminal oxygenase, ferredoxin and ferredoxin reductase components. The reduced form of the terminal oxygenase component was crystallized at 293 K by the hanging-drop vapour-diffusion method using PEG MME 550 as the precipitant under anaerobic conditions. The crystals diffracted to a resolution of 1.74 Šand belonged to space group P6(5), with unit-cell parameters a = b = 92.0, c = 243.6 Å. The asymmetric unit contained a trimer of terminal oxygenase molecules.

Leukocytapheresis is effective in inducing but not in maintaining remission in ulcerative colitis.

GOALS AND BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by dense infiltration of lymphocytes, plasma cells, neutrophils, and monocyte-macrophages into the colonic mucosa. Leukocytapheresis is a procedure for selectively removing white blood cells from withdrawn blood. It is used for the treatment of several autoimmune diseases. This study was performed to evaluate the effectiveness of leukocytapheresis for inducing and maintaining remission in corticosteroid-resistant UC, as compared with corticosteroid-responsive UC. STUDY: Forty-five patients with active UC who were treated with a dose of 1 mg/kg per day or more of prednisolone given systemically for at least 2 weeks were evaluated. Twenty patients (6 males, 14 females) in whom improvement was induced only by high doses of prednisolone were allocated as the corticosteroid-responsive group. The other 25 patients (11 males, 14 females) who did not respond to the above-mentioned dose of prednisolone therapy were allocated as the corticosteroid-resistant group and received leukocytapheresis therapy once a week for 5 weeks. Of patients who had a remission, the corticosteroid-responsive group continued to have the conventional therapy and the corticosteroid-resistant group were given leukocytapheresis once every 4 weeks for at least 2 years as maintenance therapy. RESULTS: Remission was induced by 5 weeks of leukocytapheresis in 23 of the 25 (92%) patients with corticosteroid-resistant active UC. The number of days required to achieve remission of UC was fewer in patients who received leukocytapheresis than in those who did not. Follow-up study of the patients who had remission showed similar relapse rates at 2 years in the patients who received leukocytapheresis and those given high doses of prednisolone alone. CONCLUSIONS: Leukocytapheresis is an effective treatment of acute corticosteroid-resistant UC but does not prevent the recurrence of UC.