Tertiary and quaternary structures of photoreactive Fe-type nitrile hydratase from Rhodococcus sp. N-771: roles of hydration water molecules in stabilizing the structures and the structural origin of the substrate specificity of the enzyme.

The crystal structure analysis of the Fe-type nitrile hydratase from Rhodococcus sp. N-771 revealed the unique structure of the enzyme composed of the alpha- and beta-subunits and the unprecedented structure of the non-heme iron active center [Nagashima, S., et al. (1998) Nat. Struct. Biol. 5, 347-351]. A number of hydration water molecules were identified both in the interior and at the exterior of the enzyme. The study presented here investigated the roles of the hydration water molecules in stabilizing the tertiary and the quaternary structures of the enzyme, based on the crystal structure and the results from a laser light scattering experiment for the enzyme in solution. Seventy-six hydration water molecules between the two subunits significantly contribute to the alphabeta heterodimer formation by making up the surface shape, forming extensive networks of hydrogen bonds, and moderating the surface charge of the beta-subunit. In particular, 20 hydration water molecules form the extensive networks of hydrogen bonds stabilizing the unique structure of the active center. The amino acid residues hydrogen-bonded to those hydration water molecules are highly conserved among all known nitrile hydratases and even in the homologous enzyme, thiocyanate hydrolase, suggesting the structural conservation of the water molecules in the NHase family. The crystallographic asymmetric unit contained two heterodimers connected by 50 hydration water molecules. The heterotetramer formation in crystallization was clearly explained by the concentration-dependent aggregation state of NHase found in the light scattering measurement. The measurement proved that the dimer-tetramer equilibrium shifted toward the heterotetramer dominant state in the concentration range of 10(-2)-1.0 mg/mL. In the tetramer dominant state, 50 water molecules likely glue the two heterodimers together as observed in the crystal structure. Because NHase exhibits a high abundance in bacterial cells, the result suggests that the heterotetramer is physiologically relevant. In addition, it was revealed that the substrate specificity of this enzyme, recognizing small aliphatic substrates rather than aromatic ones, came from the narrowness of the entrance channel from the bulk solvent to the active center. This finding may give a clue for changing the substrate specificity of the enzyme. Under the crystallization condition described here, one 1,4-dioxane molecule plugged the channel. Through spectroscopic and crystallographic experiments, we found that the molecule prevented the dissociation of the endogenous NO molecule from the active center even when the crystal was exposed to light.     

Effects of delayed mating on preimplantation embryos in spontaneously ovulated mice

The effects of delayed mating on mouse preimplantation embryos (78 ± 1 hours) were studied by setting up different mating periods in relation to the estimated time of spontaneous ovulation. Copulation occurred even in the late morning and early afternoon after the night of spontaneous ovulation. However, females mated in the early afternoon had no viable embryos at the time of laparotomy. Although embryonic development was not affected in the groups mated 6 or 10 hours after estimated ovulation, the percentage of degenerated embryos was increased in these groups. These results suggest that prolonged intervals between the estimated time of ovulation and mating have some deleterious effects on preimplantation embryos.     

Effect of Clostridium perfringens Beta Toxin on Blood Pressure of Rats

Guanethidine treatment or adrenal medullectomy significantly inhibited the elevation in blood pressure induced by Clostridium perfringens beta toxin, and the combination of the two drastically reduced the pressure rise, to less than 19% of that in control rats. When rats were pretreated with tetrodotoxin or hexamethonium, the toxin-evoked rise was significantly inhibited. Elevation in blood pressure induced by the toxin in spinal rats tended to be less than that in control rats. When investigated by a microscopical technique, arteriolar constriction in the mesenteric vasculature was observed after the blood pressure elevation induced by the toxin reached a maximum. Blood flow in the skin decreased with an increase in blood pressure following intravenous injection of the toxin. It is concluded that beta toxin acts on the autonomic nervous system and produces arterial constriction.     

Sex difference in susceptibility to teratogenic effect of maternal biotin deficiency in mouse embryos

When pregnant mice were fed biotin-deficient diets, cleft palate occurred more frequently in male fetuses than in female fetuses. Possible underlying mechanisms are speculated on, and some methodological problems in the analysis of sex-related differences in multiparous animals are discussed.     

Characterization of a novel acidic protein of 38 kDa, A0, in yeast ribosomes which immunologically cross-reacts with the 13 kDa acidic ribosomal proteins, A1/A2

A new ribosomal protein of 38 kDa, named A0, was detected in yeast ribosomes on immunoblotting. The antibody used here was that against A1/A2, 13 kDa acidic ribosomal proteins which cross-reacted with A0. Although A0 and A1/A2 share common antigenic determinants, they differ in the following biochemical properties. While A1/A2 could be extracted from ribosomes with ethanol and ammonium sulfate, A0 could not. A0 gave two protein spots in a less acidic region than for A1/A2 on two-dimensional gel electrophoresis. The heterogeneity observed for A0 was ascribable to phosphorylation because one spot disappeared after treatment of the ribosomes with phosphatase. The syntheses of A0 and A1/A2 are directed by different mRNA species, as judged with a cell-free translation system, ruling out the possibility that A0 is a precursor of A1/A2. Although a mammalian ribosomal protein equivalent to A0 has been shown to be associated with 13 kDa acidic proteins in the cytoplasm, essentially no A0 was detected on immunoblotting in the yeast cytosol, while a small but detectable amount of A1/A2 was present. The possibility that A0 is a eukaryotic equivalent of L10 of Escherichia coli is discussed.     

pH dependence of individual tryptophan N-1 hydrogen exchange rates in lysozyme and its chemically modified derivatives

Nuclear magnetic resonance analyses have been made of the individual hydrogen-deuterium exchange rates of tryptophan indole N-1 hydrogens in native lysozyme and its chemically modified derivatives including lysozyme with an ester cross-linkage between Glu-35 and Trp-108, lysozyme with an internal amide cross-linking between the epsilon-amino group of Lys-13 and the alpha-carboxyl group of Leu-129, and lysozyme with the beta-aspartyl sequence at Asp-101. The pH dependence curves of the exchange rates for Trp-63 and Trp-108 are different from those expected for tryptophan. The pH dependence curve for Trp-108 exchange exhibits the effects from molecular aggregation at pH above 5 and from a transition between the two conformational fluctuations at around pH 4. The exchange rates for tryptophan residues in native lysozyme and modified derivatives are not correlated with the thermodynamic or kinetic parameters in protein denaturation, suggesting that the fluctuations responsible for the exchange are not global ones. The exchange rates for tryptophan residues remote from the modification site are perturbed. Such tryptophan residues are found to be involved in a small but distinct conformational change due to the modification. Therefore, the perturbations of the N-1 hydrogen exchange rates are related to the minor change in local conformation or in conformational strain induced by the chemical modification.     

Diurnal Rhythm and Characteristics of Photosynthesis and Respiration in the Leaf and Root of a Phalaenopsis Plant

A hybrid Phalaenopsis plant was grown hydroponically on a nutrientsolution of Hyponex in the greenhouse. Typical daily patternsof CO₂ fixation, production of malic acid and citric acid andpH of the leaf and root were determined. The rate of primaryfixation of CO₂ in the leaf increased markedly in the evening,remained high until well into the afternoon, then decreasedsharply. The pattern of production of organic acids resultedfrom the fact that the rate of uptake of CO₂ gas was highestat night and fell during the day. A high correlation betweenpH value and level of malic acid in the leaves was observed,and an exponential relationship appeared to exist between theseparameters. These rhythmic sequences were not observed in theroot. The results suggest that metabolism in the leaf had characteristicsof CAM (Crassulacean Acid Metabolism), while the root did not. (Received May 6, 1988; Accepted October 25, 1988)     

Inhibitory effect of tannin on poly(ADP-ribose) glycohydrolase from human placenta

Tannin, a naturally occurring polyphenolic compound, was found to inhibit the activity of purified poly(ADP-ribose) glycohydrolase from human placenta. The inhibition was dose-dependent and half maximal with 2.8 micrograms/ml of tannin. The inhibitory effect of tannin was two and three orders of magnitude more than those of ADP-ribose and cAMP, respectively. Kinetic analysis revealed that the inhibition by tannin was competitive with respect to the substrate poly(ADP-ribose).     

Preparation and application of a fluorogenic substrate for endo-beta-xylosidase

The present paper describes a fluorometric assay for galactosaminoglycan-degrading endo-beta-xylosidase, utilizing glycosaminoglycan chains bearing a 4-methylumbelliferyl group at the reducing terminus as a substrate. This fluorogenic substrate is synthesized by human skin fibroblasts cultured in the presence of a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside. The assay is based on measurement of the fluorescence of 4-methylumbelliferone, enzymatically liberated from the synthetic substrate by endo-beta-xylosidase. We examined the applicability of the assay for analysis of endo-beta-xylosidase activity.