Crystallization and preliminary x-ray crystallographic study of NADH-cytochrome b5 reductase from pig liver microsomes

Crystals of the hydrophilic, catalytic domain (30 kDa) of pig liver NADH-cytochrome b5 reductase solubilized by the protease (cathepsin D) have been grown in a solution of polyethylene glycol by the vapor-diffusion procedure. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1) with unit-cell dimensions of a = 87.1, b = 73.2, and c = 49.0 A. The asymmetric unit contains one molecule of the enzyme. The x-ray diffraction patterns extend to 2.0-A resolution. On the other hand, the intact enzyme (35 kDa) containing the hydrophobic membrane-binding domain solubilized by the detergent (Triton N-101) has been crystallized also from the polyethylene glycol solution. The crystals are needle-shaped and still too small for x-ray diffraction study.     

Selection of Yeasts for Breadmaking by the Frozen-Dough Method

Eleven yeast strains suitable for frozen dough were selected from over 300 Saccharomyces strains. All of these were identified as Saccharomyces cerevisiae from morphological, cultural, and physiological characteristics. The selected yeast cells accumulated a higher amount of trehalose than did commercial bakers' yeast cells.     

Molecular cloning and nucleotide sequence of Thermus thermophilus HB8 trpE and trpG

The trpE gene of Thermus thermophilus HB8 was cloned by complementation of an Escherichia coli tryptophan auxotroph. The E. coli harboring the cloned gene produced the anthranilate synthase I, which was heat-stable and enzymatically active at higher temperature. The nucleotide sequence of the trpE gene and its flanking regions was determined. The trpE gene was preceded by an attenuator-like structure and followed by the trpG gene, with a short gap between them. No other gene essential for tryptophan biosynthesis was observed after the trpG gene. The amino-acid sequences of the T. themophilus anthranilate synthase I and II deduced from the nucleotide sequence were compared with those of other organisms.     

A role of heme side-chains of human hemoglobin in its function revealed by circular dichroism and resonance Raman spectroscopy

Structural changes of heme side-chains of human adult hemoglobin (Hb A) upon ligand (O₂ or CO) dissociation have been studied by circular dichroism (CD) and resonance Raman (RR) spectroscopies. We point out the occurrence of appreciable deformation of heme side-chains like vinyl and propionate groups prior to the out-of-plane displacement of heme iron. Referring to the recent fine resolved crystal structure of Hb A, the deformations of heme side-chains take place only in the β subunits. However, these changes are not observed in the isolated β chain (β₄ homotetramer) and, therefore, are associated with the α–β inter-subunit interactions. For the communications between α and β subunits in Hb A regarding signals of ligand dissociation, possible routes are proposed on the basis of the time-resolved absorption, CD, MCD (magnetic CD), and RR spectroscopies. Our finding of the movements of heme side-chains would serve as one of the clues to solve the cooperative O₂ binding mechanism of Hb A.     

Discovery of NR2B-selective antagonists via scaffold hopping and pharmacokinetic profile optimization

Selective N-methyl-d-aspartate receptor subunit 2B (NR2B) antagonists show potential as analgesic drugs, and do not cause side effects associated with non-selective N-methyl-d-aspartate (NMDA) antagonists. Using a scaffold-hopping approach, we previously identified isoxazole derivative 4 as a potent selective NR2B antagonist. In this study, further scaffold hopping of isoxazole derivative 4 and optimization of its pharmacokinetic profile led to the discovery of the orally bioavailable compound 6v. In a rat study of analgesia, 6v demonstrated analgesic effects against neuropathic pain.     

Pyrrolinone derivatives as a new class of P2X3 receptor antagonists Part 2: Discovery of orally bioavailable compounds

Some P2X3 receptor antagonists have been developed as new therapeutic drugs for pain. We discovered a novel chemotype of P2X3 receptor antagonists with a pyrrolinone skeleton. Because of SAR studies to improve bioavailability of lead compound 2, compound (R)-24 was identified, which showed an analgesic effect against neuropathic pain by oral administration. We constructed a human P2X3 homology model as a template for the zebrafish P2X4 receptor, which agreed with SAR studies of pyrrolinone derivatives.     

Identification of the gene encoding a type 1 RNase H with an N-terminal double-stranded RNA binding domain from a psychrotrophic bacterium

The gene encoding a bacterial type 1 RNase H, termed RBD-RNase HI, was cloned from the psychrotrophic bacterium Shewanella sp. SIB1, overproduced in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RBD-RNase HI consists of 262 amino acid residues and shows amino acid sequence identities of 26% to SIB1 RNase HI, 17% to E. coli RNase HI, and 32% to human RNase H1. SIB1 RBD-RNase HI has a double-stranded RNA binding domain (RBD) at the N-terminus, which is commonly present at the N-termini of eukaryotic type 1 RNases H. Gel mobility shift assay indicated that this domain binds to an RNA/DNA hybrid in an isolated form, suggesting that this domain is involved in substrate binding. SIB1 RBD-RNase HI exhibited the enzymatic activity both in vitro and in vivo. Its optimum pH and metal ion requirement were similar to those of SIB1 RNase HI, E. coli RNase HI, and human RNase H1. The specific activity of SIB1 RBD-RNase HI was comparable to that of E. coli RNase HI and was much higher than those of SIB1 RNase HI and human RNase H1. SIB1 RBD-RNase HI showed poor cleavage-site specificity for oligomeric substrates. SIB1 RBD-RNase HI was less stable than E. coli RNase HI but was as stable as human RNase H1. Database searches indicate that several bacteria and archaea contain an RBD-RNase HI. This is the first report on the biochemical characterization of RBD-RNase HI.     

Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach

The gene encoding a cutinase homolog, LC-cutinase, was cloned from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. LC-cutinase shows the highest amino acid sequence identity of 59.7% to Thermomonospora curvata lipase. It also shows the 57.4% identity to Thermobifida fusca cutinase. When LC-cutinase without a putative signal peptide was secreted to the periplasm of Escherichia coli cells with the assistance of the pelB leader sequence, more than 50% of the recombinant protein, termed LC-cutinase*, was excreted into the extracellular medium. It was purified and characterized. LC-cutinase* hydrolyzed various fatty acid monoesters with acyl chain lengths of 2 to 18, with a preference for short-chain substrates (C(4) substrate at most) most optimally at pH 8.5 and 50°C, but could not hydrolyze olive oil. It lost activity with half-lives of 40 min at 70°C and 7 min at 80°C. LC-cutinase* had an ability to degrade poly(ε-caprolactone) and polyethylene terephthalate (PET). The specific PET-degrading activity of LC-cutinase* was determined to be 12 mg/h/mg of enzyme (2.7 mg/h/μkat of pNP-butyrate-degrading activity) at pH 8.0 and 50°C. This activity is higher than those of the bacterial and fungal cutinases reported thus far, suggesting that LC-cutinase* not only serves as a good model for understanding the molecular mechanism of PET-degrading enzyme but also is potentially applicable for surface modification and degradation of PET.     

Taxonomic study of the Burmoniscus ocellatus complex (Crustacea, Isopoda, Oniscidea) in Japan shows genetic diversification in the southern Ryukyus, southwestern Japan

To clarify the taxonomic status of the Burmoniscus ocellatus complex in Japan, we carried out morphological observations and phylogenetic analyses of specimens collected from Yonagunijima, Iriomotejima, Ishigakijima, and Miyakojima Islands of the southern Ryukyus and from Okinawajima Island of the central Ryukyus in southwestern Japan. Observations of the holotypes of Aphiloscia iriomotensis ( Nunomura, 1986 ), Ap. ishigakiensis ( Nunomura, 1986 ), and Ap. yonakuniensis ( Nunomura, 1986 ), in addition to the specimens newly collected from the five islands, indicated that these specimens belong to the genus Burmoniscus. Analyses of five morphological characters of 268 specimens collected from the five islands showed that the body size of Okinawajima specimens is distinctly smaller than those of the specimens from the southern Ryukyus. The ranges of the five morphological characters tended to overlap among the specimens from Yonagunijima, Iriomotejima, Ishigakijima and Miyakojima Islands; these morphological characters were congruent with those of 6. ocellatus (Verhoeff, 1928). The phylogenetic analyses were based on three regions of mitochondrial DNA-COI, 12S rRNA, and 16S rRNA-and indicated that the specimens collected from the southern Ryukyus constitute a monophyletic group, which is clearly distinct from the clade composed of the Okinawajima specimens. These results strongly suggest that Ap. iriomotensis, Ap. ishigakiensis, and Ap. yonakuniensis are synonymous with B. ocellatus, a species widely distributed in the southern Ryukyus. On the other hand, the species from Okinawajima Island in the central Ryukyus is considered to be an undescribed Burmoniscus species, which is closely related to but clearly distinct from S. ocellatus.