Involvement of C-terminal amino acids of a hyperthermophilic serine racemase in its thermostability.

Pyrobaculum islandicum is a hyperthermophilic archaeon that grows optimally at 95-100 °C. In the previous study, we extensively purified a serine racemase from this organism and cloned the gene for overexpression in Escherichia coli (Ohnishi et al. 2008). This enzyme also exhibits highly thermostable L-serine/L-threonine dehydratase activity. In the present study, we aimed to elucidate the molecular mechanisms underlying the high thermostability of this enzyme. A recombinant variant of this enzyme, PiSRvt, constructed by truncating the C-terminal 72 amino acids, was compared with the native enzyme, PiSR. The dehydratase activity of PiSR and PiSRvt was found to owe to a homotrimer and a monomer, respectively, that demonstrated high and moderate thermostability, respectively. These observations reveal that the C-terminal region contributes to monomer trimerization that provides the extreme thermostability.

Structure of pyridoxal 5'-phosphate-bound D-threonine aldolase from Chlamydomonas reinhardtii.

D-Threonine aldolase (DTA) is a pyridoxal-5'-phosphate-dependent enzyme which catalyzes the reversible aldol reaction of glycine with a corresponding aldehyde to yield the D-form ß-hydroxy-α-amino acid. This study produced and investigated the crystal structure of DTA from Chlamydomonas reinhardtii (CrDTA) at 1.85 Šresolution. To our knowledge, this is the first report on the crystal structure of eukaryotic DTA. Compared with the structure of bacterial DTA, CrDTA has a similar arrangement of active-site residues. On the other hand, we speculated that some non-conserved residues alter the affinity for substrates and inhibitors. The structure of CrDTA could provide insights into the structural framework for structure-guided protein engineering studies to modify reaction selectivity.

Amino Acid Conjugates of ent-Beyerane-type Diterpenoids and Oleanane-type Triterpenoids: Synthesis and Evaluation of Cytotoxic Activities.

The aim of this study was to increase the cytotoxic activities of terpenoids via amino acid conjugation. Thus, 21 new ester derivatives (5-15, 19-27, and 29) were prepared by conjugation of the hydroxy groups in ent-beyerane-type diterpenoids (4) and oleanane-type triterpenoids (18), and their cytotoxic activity against three human cancer cell lines (leukemia, lung, and stomach) were evaluated. The prepared compounds showed cytotoxic effects; in particular, all amino acid conjugates of ent-beyerane-type diterpenoids (5-13) exhibited potent cytotoxic activity (IC50 1.0-3.7 µM for HL60, 1.7-8.2 µM for A549, and 2.5-11.7 µM for MKN45). In addition, no differences were observed in the cytotoxic activities of l- and d-type amino acid conjugates.

Rapid removal of detergent in glycolipids using ionic liquids.

Detergent removal in glycolipid after sample preparation, such as enzymatic reaction or isolation of detergent-resistant membrane microdomain, is indispensable for further structural characterization. We previously established the rapid and effective method of detergent removal in glycolipid samples from glass test tube using 1,2-dichloroethane (DCE) washing. However, the use of DCE has several drawbacks, such as environmental risks, harmful effects (potentially carcinogenic), and high vaporability and flammability. To solve the issue, we used ionic liquids to remove detergents from glycolipid samples, and found 1-butyl-3-methylimidazolium iodide was a suitable alternative for DCE.

Purification of Helicobacter pylori NCTC 11637 cytochrome bc1 and respiration with D-proline as a substrate.

Helicobacter pylori is a microaerophilic bacterium associated with gastric inflammation and peptic ulcers. Knowledge of how pathogenic organisms produce energy is important from a therapeutic point of view. We found d-amino acid dehydrogenase-mediated electron transport from d-proline or d-alanine to oxygen via the respiratory chain in H. pylori. Coupling of the electron transport to ATP synthesis was confirmed by using uncoupler reagents. We reconstituted the electron transport chain to demonstrate the electron flow from the d-amino acids to oxygen using the recombinant cytochrome bc(1) complex, cytochrome c-553, and the terminal oxidase cytochrome cbb(3) complex. Upon addition of the recombinant d-amino acid dehydrogenase and d-proline or d-alanine to the reconstituted electron transport system, reduction of cytochrome cbb(3) and oxygen consumption was revealed spectrophotometrically and polarographically, respectively. Among the constituents of H. pylori's electron transport chain, only the cytochrome bc(1) complex had been remained unpurified. Therefore, we cloned and sequenced the H. pylori NCTC 11637 cytochrome bc(1) gene clusters encoding Rieske Fe-S protein, cytochrome b, and cytochrome c(1), with calculated molecular masses of 18 kDa, 47 kDa, and 32 kDa, respectively, and purified the recombinant monomeric protein complex with a molecular mass of 110 kDa by gel filtration. The absorption spectrum of the recombinant cytochrome bc(1) complex showed an alpha peak at 561 nm with a shoulder at 552 nm.

D-Amino acid dehydrogenase from Helicobacter pylori NCTC 11637.

Helicobacter pylori is a microaerophilic bacterium, associated with gastric inflammation and peptic ulcers. D-Amino acid dehydrogenase is a flavoenzyme that digests free neutral D-amino acids yielding corresponding 2-oxo acids and hydrogen. We sequenced the H. pylori NCTC 11637 D-amino acid dehydrogenase gene, dadA. The primary structure deduced from the gene showed low similarity with other bacterial D-amino acid dehydrogenases. We purified the enzyme to homogeneity from recombinant Escherichia coli cells by cloning dadA. The recombinant protein, DadA, with 44 kDa molecular mass, possessed FAD as cofactor, and showed the highest activity to D-proline. The enzyme mediated electron transport from D-proline to coenzyme Q(1), thus distinguishing it from D-amino acid oxidase. The apparent K(m) and V(max) values were 40.2 mM and 25.0 micromol min(-1) mg(-1), respectively, for dehydrogenation of D-proline, and were 8.2 microM and 12.3 micromol min(-1) mg(-1), respectively, for reduction of Q(1). The respective pH and temperature optima were 8.0 and 37 degrees C. Enzyme activity was inhibited markedly by benzoate, and moderately by SH reagents. DadA showed more similarity with mammalian D-amino acid oxidase than other bacterial D-amino acid dehydrogenases in some enzymatic characteristics. Electron transport from D-proline to a c-type cytochrome was suggested spectrophotometrically.

Crystallization and X-ray analysis of D-threonine aldolase from Chlamydomonas reinhardtii.

D-Threonine aldolase from the green alga Chlamydomonas reinhardtii (CrDTA) catalyzes the interconversion of several ß-hydroxy-D-amino acids (e.g. D-threonine) and glycine plus the corresponding aldehydes. Recombinant CrDTA was overexpressed in Escherichia coli and purified to homogeneity; it was subsequently crystallized using the hanging-drop vapour-diffusion method at 295 K. Data were collected and processed at 1.85 Šresolution. Analysis of the diffraction pattern showed that the crystal belonged to space group P1, with unit-cell parameters a = 64.79, b = 74.10, c = 89.94 Å, α = 77.07, ß = 69.34, γ = 71.93°. The asymmetric unit contained four molecules of CrDTA. The Matthews coefficient was calculated to be 2.12 Å3 Da-1 and the solvent content was 41.9%.

Cloning and characterization of d-threonine aldolase from the green alga Chlamydomonas reinhardtii.

d-Threonine aldolase (DTA) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent interconversion of d-threonine and glycine plus acetaldehyde. The enzyme is a powerful tool for the stereospecific synthesis of various ß-hydroxy amino acids in synthetic organic chemistry. In this study, DTA from the green alga Chlamydomonas reinhardtii was discovered and characterized, representing the first report to describe the existence of eukaryotic DTA. DTA was overexpressed in recombinant Escherichia coli BL21 (DE3) cells; the specific activity of the enzyme in the cell-free extract was 0.8 U/mg. The recombinant enzyme was purified to homogeneity by ammonium sulfate fractionation, DEAE-Sepharose, and Mono Q column chromatographies (purified enzyme 7.0 U/mg). For the cleavage reaction, the optimal temperature and pH were 70 °C and pH 8.4, respectively. The enzyme demonstrated 90% of residual activity at 50 °C for 1 h. The enzyme catalyzed the synthesis of d- and d-allo threonine from a mixture of glycine and acetaldehyde (the diastereomer excess of d-threonine was 18%). DTA was activated by several divalent metal ions, including manganese, and was inhibited by PLP enzyme inhibitors and metalloenzyme inhibitors.

The presence of high concentrations of free D-amino acids in human saliva.

Free neutral D-amino acids have previously been detected in human plasma, usually accounting for less than 2% of the total free amino acid concentration (D-amino acid ratio) [Nagata, Y., Masui, R., Akino, T., 1992a. The presence of free D-serine, D-alanine and D-proline in human plasma. Experientia 48, 986-988. Nagata, Y., Yamamoto, K., Shimojo, T., 1992b. Determination of D- and L-amino acids in mouse kidney by high-performance liquid chromatography. Journal of Chromatography 575, 147-152. Nagata, Y., Yamamoto, K., Shimojo, T., Konno, R., Yasumura, Y., Akino, T., 1992c. The presence of free D-alanine, D-proline and D-serine in mice. Biochimca et Biiophysica Acta 1115, 208-211]. In the present study to search for the source of free D-amino acids, D- and L-enantiomers of the major non-essential amino acids, i.e., the free form of serine, alanine, proline, aspartate and glutamate were analyzed by HPLC in human saliva, submandibular glands and oral epithelial cells. The D-enantiomer ratios to total of free alanine or proline were 35% and 20%, respectively, in saliva. The ratios of the other D-amino acids were less than 11%. The effect of ingested food and oral bacteria on the saliva amino acid levels was suggested to be insignificant. D-Alanine and d-aspartate were also detected in the submandibular gland in ratios up to 5%, and D-alanine and d-proline were found in oral epithelial cells in ratios of 18% and 5%, respectively. The submandibular gland and oral epithelial cells are suggested to be possible sources of the saliva D-alanine and D-aspartate.

Participation of D-serine in the development and reproduction of the silkworm Bombyx mori.

The silkworm Bombyx mori contains high concentrations of free D-serine, an optical isomer of L-serine. To elucidate its function, we first investigated the localization of D-serine in various organs of silkworm larvae, pupae, and adult moths. Using immunohistochemical analysis with an anti-D-serine antibody, we found D-serine in the microvilli of midgut goblet and cylindrical cells and in peripheral matrix components of testicular and ovarian cells. By spectrophotometric analysis, D-serine was also found in the hemolymph and fat body. D-Alanine was not detected in the various organs by immunohistochemistry. Serine racemase, which catalyzes the inter-conversion of L- and D-serine, was found to co-localize with D-serine, and D-serine production from L-serine by intrinsic serine racemase was suggested. O-Phospho-L-serine is an inhibitor of serine racemase, and it was administered to the larvae to reduce the D-serine level. This reagent decreased the midgut caspase-3 level and caused a delay in spermatogenesis and oogenesis. The reagent also decreased mature sperm and egg numbers, suggesting D-serine participation in these processes. D-Serine administration induced an increase in pyruvate levels in testis, midgut, and fat body, indicating conversion of D-serine to pyruvate. On the basis of these results, together with our previous investigation of ATP biosynthesis in testis, we consider the possible involvement of D-serine in ATP synthesis for metamorphosis and reproduction.

Purification of Pyridylaminated Oligosaccharides Using 1,2-Dichloroethane Extraction.

Fluorescence derivatization of the oligosaccharides released from glycoconjugates is widely used for precise structural characterization. To ensure labeling of the oligosaccharides, a large excess of fluorescence reagents is usually added to the reaction tube. Therefore, any excess reagents and by-products of the labeling reaction should be removed by several column chromatographies, including using a cellulose cartridge or spin columns. However, these purification steps are often time-consuming, expensive, and laborious. In this study, we found that 1,2-dichloroethane extraction could effectively and easily purify pyridylaminated oligosaccharides with a high recovery rate.

Methemoglobin reduction mediated by D-amino acid dehydrogenase in Propsilocerus akamusi (Tokunaga) larvae.

A methemoglobin (metHb) reduction system is required for aerobic respiration. In humans, Fe(III)-heme-bearing metHb (the oxidized form of hemoglobin), which cannot bind oxygen, is converted to Fe(II)-heme-bearing oxyhemoglobin (oxyHb, the reduced form), which can bind oxygen, in a system comprising NADH, NADH-cytochrome b5 reductase, and cytochrome b5. However, the mechanism of metHb reduction in organisms that inhabit oxygen-deficient environments is unknown. In the coelomic fluid of the larvae of Propsilocerus akamusi, which inhabit a microaerobic environment, we found that metHb was reduced by D-alanine. We purified an FAD-containing enzyme, D-amino acid dehydrogenase (DAD), and component V hemoglobin from the larvae. Using the purified components and spectrophotometric analyses, we showed a novel function of DAD: DAD-mediation of P. akamusi component V metHb reduction with using D-alanine as an electron donor. P. akamusi larvae possess this D-alanine-DAD metHb reduction system in addition to a previously discovered NADH-NADH-cytochrome b5 reductase system. This is the first report of the presence of DAD in a multicellular organism. The molecular mass of DAD was estimated to be 45 kDa. The optimal pH and temperature of the enzyme were 7.4 and 20 °C, respectively, and the optimal substrate was D-alanine. The enzyme activity was inhibited by benzoate and sulfhydryl-binding reagents.

Methemoglobin reduction by NADH-cytochrome b(5) reductase in Propsilocerus akamusi larvae.

For oxygen respiration, a methemoglobin (metHb) reduction system is needed in the cell because metHb cannot bind oxygen. We examined the insect Propsilocerus akamusi larvae to elucidate the metHb reduction system in an organism that inhabits an oxygen-deficient environment. NADH-dependent reduction of metHb in coelomic fluid suggested the coexistence of cytochrome b5 reductase (b5R) and cytochrome b5 with hemoglobin in the fluid and that these proteins were involved in physiological metHb reduction in the larvae. The presence of b5R was revealed by purifying b5R to homogeneity from the midge larvae. Using purified components, we showed that larval metHb was reduced via the NADH-b5R (FAD)-cytochrome b5-metHb pathway, a finding consistent with that in aerobic vertebrates, specifically humans and rabbits, and b5R function between mammal and insect was conserved. b5R was identified as a monomeric FAD-containing enzyme; it had a molecular mass of 33.2 kDa in gel-filtration chromatography and approximately 37 kDa in SDS-PAGE analysis. The enzyme's optimal pH and temperature were 6.4 and 25 °C, respectively. The apparent Km and Vmax values were 345 µM and 160 µmol min(-1) mg(-1), respectively, for ferricyanide and 328 µM and 500 µmol min(-1) mg(-1), respectively, for 2,6-dichlorophenolindophenol. The enzyme reaction was inhibited by benzoate, p-hydroxymercuribenzoate, iodoacetamide, and iodoacetate, and was not inhibited by metal ions or EDTA.

Corrosion by bacteria of concrete in sewerage systems and inhibitory effects of formates on their growth.

Not only sulfur-oxidizing bacteria but also an acidophilic iron-oxidizing bacterium (or bacteria) were found in the corroded concrete from several sewerage systems in Japan. The surface pH of concrete test piece exposed to an atmosphere containing hydrogen sulfide of the concentrations more than 600 ppm in the systems was usually below 2 after a month. This was attributable to ability of the sulfur-oxidizing bacteria to grow in the thin water layer which contained hydrogen sulfide and covered the piece even when the surface pH of concrete was 12-13. When the sulfuroxidizing bacteria grew in the surface of concrete and produced sulfuric acid, the pH of the inner parts of concrete was lowered where the bacteria were hardly found. Probably, sulfuric acid formed by the bacteria in the surface parts penetrated into the inner parts. The different species of sulfur-oxidizing bacteria were found in different sewerage systems. The growth of the sulfur-oxidizing and acidophilic iron-oxidizing bacteria was completely inhibited by formates, especially by calcium formate of concentrations more than 50 mM. Calcium formate can protect concrete in sewerage systems from bacterial corrosion.

Effect of D-serine on spermatogenesis and extracellular signal-regulated protein kinase (ERK) phosphorylation in the testis of the silkworm, Bombyx mori.

Although the pupae and larvae of Bombyx mori possess especially large amounts of free d-serine, the physiological role of the amino acid in the silkworm is unknown. We investigated the effect of d-serine on spermatogenesis. A lowered d-serine level throughout larval development caused a delay in spermatogenesis and resulted in reduced numbers of eupyrene sperm. Administration of d-serine transiently increased the activation of extracellular signal-regulated protein kinase1/2 (ERK1/2; hereafter, ERK) by approximately 25% in the testis of day 3 fifth instar larvae. l-Serine had no effect on ERK activation, and other organs did not respond to d-serine. The effect of d-serine on ERK activation was confirmed by administering d-serine dehydratase, an enzyme that specifically degrades d-serine, and the enzyme's inhibitor, hydroxylamine. ERK phosphorylation in the testis was significantly inhibited by Go6983 and U0126, inhibitors of protein kinase C (PKC) and mitogen-associated protein kinase kinase 1/2 (MEK), respectively, but not by H-89, a protein kinase A (PKA) inhibitor, indicating that ERK was activated in the testis via PKC and MEK but not via PKA. The inhibition of ERK phosphorylation by Go6983 or U0126 was reduced by 20-30% by d-serine. Roughly 30% of c-Raf phosphorylation at an inhibitory site (Ser259) was decreased by the addition of d-serine. These results suggest that d-serine activates ERK in the testis of silkworms through a pathway including c-Raf but not PKC or MEK. Immunohistochemistry confirmed d-serine-induced ERK phosphorylation in the testis and revealed the presence of phospho-ERK in the nuclei of spermatocytes and spermatids.

A case of acute posterior multifocal placoid pigment epitheliopathy demonstrating vogt-koyanagi-harada disease-like optical coherence tomography findings in the acute stage.

PURPOSE: We report a case of acute posterior multifocal placoid pigment epitheliopathy (APMPPE), which was difficult to differentiate from posterior pole-type Vogt-Koyanagi-Harada (VKH) disease because the lesions were mainly located in the macula bilaterally. CASE REPORT: A 33-year-old man presented with rapid bilateral loss of vision. Fundoscopy revealed yellow-white subretinal lesions in the posterior pole of both eyes. Optical coherence tomography (OCT) revealed the presence of subretinal fluid with a subretinal septum. After initiation of systemic steroids, OCT revealed that the amount of subretinal fluid decreased immediately. However, vision loss was less responsive to the therapy, and OCT revealed partial reorganization of the inner segment/outer segment (IS/OS) line in the bilateral macular areas after therapy. DISCUSSION: In our case, the location of the macular lesions made it difficult to differentiate APMPPE from VKH disease by fluorescein angiography. OCT images showed VKH disease-like findings of serous retinal detachment with a subretinal septum. The outer nuclear layer disappeared and the IS/OS line in the affected area was disorganized in the acute stage of the disease. In this case, the rapid loss of vision was specific to the onset pattern of APMPPE, and the slow response to therapy was very different from the response typically observed in VKH disease. Thus, careful consideration of the clinical course is important for diagnosing APMPPE.

D-Amino acid oxidase and presence of D-proline in Xenopus laevis.

We purified D-amino acid oxidase (EC 1.4.3.3, DAO) from Xenopus laevis tadpoles. The optimal temperature and pH for enzyme activity were 35-40 °C and 8.3-9.0, respectively, depending on the substrate amino acids available to the enzyme; the highest activity was observed with D-proline followed by D-phenylalanine. Activity was significantly inhibited by p-hydroxymercuribenzoate, but only moderately by p-chloromercuribenzoate or benzoate. Enzyme activity was increased until the final tadpole stage, but was reduced to one-third in the adult and was localized primarily in the kidney. The tadpoles contained high concentrations of D-proline close to the final developmental stage and nearly no D-amino acids were detected in the adult frog, indicating that D-amino acid oxidase functions in metamorphosis.

Choroidal folds in acute-stage vogt-koyanagi-harada disease patients with relatively short axial length.

PURPOSE: To report 2 cases of Vogt-Koyanagi-Harada disease accompanied by remarkable choroidal folds in the acute stage. The early indicator of recurrence in these 2 cases was the identification of choroidal folds by spectral-domain optical coherence tomography (SD-OCT). CASE REPORTS: A 68-year-old woman (Case 1) presented with visual loss in both eyes. Funduscopic examination revealed optic disc swelling and serous retinal detachment in both eyes. SD-OCT revealed remarkable choroidal folds and serous retinal detachment. After the initiation of systemic steroid treatment, choroidal folds disappeared rapidly and the amount of serous retinal detachment reduced remarkably. Choroidal folds observed on SD-OCT were the early indicators of recurrence prior to the emergence of serous retinal detachment. A 62-year-old woman (Case 2) presented with bilateral blurred vision and metamorphopsia. SD-OCT showed remarkable choroidal folds and serous retinal detachment in both eyes. After the initiation of systemic steroid treatment, choroidal folds and serous retinal detachment disappeared. At the time of recurrence, choroidal folds were observed by OCT. DISCUSSION: During monitoring of Vogt-Koyanagi-Harada disease treatment, choroidal folds could be an early sign of recurrence. When choroidal folds are observed and recognized as an early indicator of recurrence, a prompt increase in steroids can improve the patients' prognosis. Finally, both cases presented here had relatively short axial lengths, and we speculate that a shortened axial length may be a cause of choroidal folds in the acute stage of the disease.