Overexpression and characterization of Escherichia coli dihydropyrimidine dehydrogenase: a four iron-sulphur cluster containing flavoprotein.

Escherichia coli dihydropyrimidine dehydrogenase (EcDPD) catalyses the NADH-dependent reduction of uracil and thymine to the corresponding 5,6-dihydropyrimidines to control their metabolite pools. EcDPD consists of two subunits, PreT and PreA, and requires FAD, FMN and Fe-S clusters for activity. Recombinant EcDPD with a C-terminal His6-tagged-PreA subunit was overproduced in a DPD-lacking E. coli cells with augmented Fe-S cluster synthesis. Anaerobic purification resulted in purified enzyme with a specific activity of 13 µmol min-1 mg-1. The purified EcDPD was a heterotetramer and contained 0.81 FAD, 0.99 FMN, 14 acid-labile sulphur and 15 iron per PreT-PreA dimer. The enzyme exhibited Michaelis-Menten kinetics for both the forward and reverse reactions, which is distinct from mammalian DPDs showing substrate inhibition kinetics. For uracil reduction, the kcat, kcat/KNADH and kcat/Kuracil values were constant over the pH range of 5.5-10. For dihydrouracil (DHU) dehydrogenation, the pH-dependence of the kcat and kcat/KNAD+ values indicated that a residue with a pKa of 6.6 must be deprotonated for activity. Biochemical and kinetic comparisons with pig DPD revealed that protonation sates of the catalytically competent forms of EcDPD are distinct from those of pig enzyme.

Exopolysaccharides from a Scandinavian fermented milk viili increase butyric acid and Muribaculum members in the mouse gut.

Starter culture of viili contains lactic acid bacteria belonging to Lactococcus lactis. These bacteria secrete large polysaccharides (EPSs) into milk, resulting in a ropy texture of viili. In mouse experiments, a large dose of EPS (5-140 mg/day) has been shown to alleviate severity of artificially induced illness through modulation of the gut microbiota. The present study investigated whether supplementary amounts of EPS affects the gut microbiota of normal mouse. EPS with high glucosamine content (VEPS) was isolated from home-made viili. C57BL/6J male mice fed ordinary diet took 49 ± 1 µg VEPS/day for 28 days by drinking ad libitum tap water containing 8 µg/mL VEPS. The relative abundance of Muribaculum increased significantly by VEPS supplementation. The relative abundance of fecal butyric acid decreased in control mice, and VEPS prevented this decrease. These findings indicated that the gut microbiota can be modulated by a small dose of VEPS.

Structural insights into the catalytic mechanism of cysteine (hydroxyl) lyase from the hydrogen sulfide-producing oral pathogen, Fusobacterium nucleatum.

Hydrogen sulfide (H2S) plays important roles in the pathogenesis of periodontitis. Oral pathogens typically produce H2S from l-cysteine in addition to pyruvate and [Formula: see text] However, fn1055 from Fusobacterium nucleatum subsp. nucleatum ATCC 25586 encodes a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the production of H2S and l-serine from l-cysteine and H2O, an unusual cysteine (hydroxyl) lyase reaction (ß-replacement reaction). To reveal the reaction mechanism, the crystal structure of substrate-free Fn1055 was determined. Based on this structure, a model of the l-cysteine-PLP Schiff base suggested that the thiol group forms hydrogen bonds with Asp232 and Ser74, and the substrate α-carboxylate interacts with Thr73 and Gln147 Asp232 is a unique residue to Fn1055 and its substitution to asparagine (D232N) resulted in almost complete loss of ß-replacement activity. The D232N structure obtained in the presence of l-cysteine contained the α-aminoacrylate-PLP Schiff base in the active site, indicating that Asp232 is essential for the addition of water to the α-aminoacrylate to produce the l-serine-PLP Schiff base. Rapid-scan stopped-flow kinetic analyses showed an accumulation of the α-aminoacrylate intermediate during the reaction cycle, suggesting that water addition mediated by Asp232 is the rate-limiting step. In contrast, mutants containing substitutions of other active-site residues (Ser74, Thr73, and Gln147) exhibited reduced ß-replacement activity by more than 100-fold. Finally, based on the structural and biochemical analyses, we propose a mechanism of the cysteine (hydroxyl) lyase reaction by Fn1055. The present study leads to elucidation of the H2S-producing mechanism in F. nucleatum.

The identification of á´ -tryptophan as a bioactive substance for postembryonic ovarian development in the planarian Dugesia ryukyuensis.

Many metazoans start germ cell development during embryogenesis, while some metazoans possessing pluripotent stem cells undergo postembryonic germ cell development. The latter reproduce asexually but develop germ cells from pluripotent stem cells or dormant primordial germ cells when they reproduce sexually. Sexual induction of the planarian Dugesia ryukyuensis is an important model for postembryonic germ cell development. In this experimental system, hermaphroditic reproductive organs are differentiated in presumptive gonadal regions by the administration of a crude extract from sexual planarians to asexual ones. However, the substances involved in the first event during postembryonic germ cell development, i.e., ovarian development, remain unknown. Here, we aimed to identify a bioactive compound associated with postembryonic ovarian development. Bioassay-guided fractionation identified ʟ-tryptophan (Trp) on the basis of electrospray ionization-mass spectrometry, circular dichroism, and nuclear magnetic resonance spectroscopy. Originally masked by a large amount of ʟ-Trp, ᴅ-Trp was detected by reverse-phase high-performance liquid chromatography. The ovary-inducing activity of ᴅ-Trp was 500 times more potent than that of ʟ-Trp. This is the first report describing a role for an intrinsic ᴅ-amino acid in postembryonic germ cell development. Our findings provide a novel insight into the mechanisms of germ cell development regulated by low-molecular weight bioactive compounds.

Novel Therapeutic Role for Dipeptidyl Peptidase III in the Treatment of Hypertension.

Dipeptidyl peptidase III (DPP III) cleaves dipeptide residues from the N terminus of polypeptides ranging from 3 to 10 amino acids in length and is implicated in pathophysiological processes through the breakdown of certain oligopeptides or their fragments. In this study, we newly identified the biochemical properties of DPP III for angiotensin II (Ang II), which consists of 8 amino acids. DPP III quickly and effectively digested Ang II with Km = 3.7×10(-6) mol/L. In the in vivo experiments, DPP III remarkably reduced blood pressure in Ang II-infused hypertensive mice without alteration of heart rate. DPP III did not affect hemodynamics in noradrenalin-induced hypertensive mice or normotensive mice, suggesting specificity for Ang II. When DPP III was intravenously injected every other day for 4 weeks after Ang II osmotic minipump implantation in mice, Ang II-induced cardiac fibrosis and hypertrophy were significantly attenuated. This DPP III effect was at least similar to that caused by an angiotensin receptor blocker candesartan. Furthermore, administration of DPP III dramatically reduced the increase in urine albumin excretion and kidney injury and inflammation markers caused by Ang II infusion. Both DPP III and candesartan administration showed slight additive inhibition in the albumin excretion. These results reveal a novel potential use of DPP III in the treatment of hypertension and its protective effects on hypertension-sensitive organs, such as the heart and kidneys.

The Lipid Kinase PI5P4Kß Is an Intracellular GTP Sensor for Metabolism and Tumorigenesis.

While cellular GTP concentration dramatically changes in response to an organism's cellular status, whether it serves as a metabolic cue for biological signaling remains elusive due to the lack of molecular identification of GTP sensors. Here we report that PI5P4Kß, a phosphoinositide kinase that regulates PI(5)P levels, detects GTP concentration and converts them into lipid second messenger signaling. Biochemical analyses show that PI5P4Kß preferentially utilizes GTP, rather than ATP, for PI(5)P phosphorylation, and its activity reflects changes in direct proportion to the physiological GTP concentration. Structural and biological analyses reveal that the GTP-sensing activity of PI5P4Kß is critical for metabolic adaptation and tumorigenesis. These results demonstrate that PI5P4Kß is the missing GTP sensor and that GTP concentration functions as a metabolic cue via PI5P4Kß. The critical role of the GTP-sensing activity of PI5P4Kß in cancer signifies this lipid kinase as a cancer therapeutic target.

Immunohistochemical localization of D-serine dehydratase in chicken tissues.

Chicken D-serine dehydratase (DSD) degrades d-serine to pyruvate and ammonia. The enzyme requires both pyridoxal 5'-phosphate and Zn(2+) for its activity. d-Serine is a physiological coagonist that regulates the activity of the N-methyl-d-aspartate receptor (NMDAR) for l-glutamate. We have recently found in chickens that d-serine is degraded only by DSD in the brain, whereas it is also degraded to 3-hydroxypyruvate by d-amino acid oxidase (DAO) in the kidney and liver. In mammalian brains, d-serine is degraded only by DAO. It has not been clarified why chickens selectively use DSD for the control of d-serine concentrations in the brain. In the present study, we measured DSD activity in chicken tissues, and examined the cellular localization of DSD using a specific anti-chicken DSD antibody. The highest activity was found in kidney. Skeletal muscles and heart showed no activity. In chicken brain, cerebellum showed about 6-fold-higher activity (1.1 ± 0.3 U/g protein) than cerebrum (0.19 ± 0.03 U/g protein). At the cellular level DSD was demonstrated in proximal tubule cells of the kidney, in hepatocytes, in Bergmann-glia cells of the cerebellum and in astrocytes. The finding of DSD in glial cells seems to be important because d-serine is involved in NMDAR-dependent brain functions.

Planarian D-amino acid oxidase is involved in ovarian development during sexual induction.

To elucidate the molecular mechanisms underlying switching from asexual to sexual reproduction, namely sexual induction, we developed an assay system for sexual induction in the hermaphroditic planarian species Dugesia ryukyuensis. Ovarian development is the initial and essential step in sexual induction, and it is followed by the formation of other reproductive organs, including the testes. Here, we report a function of a planarian D-amino acid oxidase, Dr-DAO, in the control of ovarian development in planarians. Asexual worms showed significantly more widespread expression of Dr-DAO in the parenchymal space than did sexual worms. Inhibition of Dr-DAO by RNAi caused the formation of immature ovaries. In addition, we found that feeding asexual worms 5 specific D-amino acids could induce the formation of immature ovaries that are similar to those observed in Dr-DAO knockdown worms, suggesting that Dr-DAO inhibits the formation of immature ovaries by degrading these D-amino acids. Following sexual induction, Dr-DAO expression was observed in the ovaries. The knockdown of Dr-DAO during sexual induction delayed the maturation of the other reproductive organs, as well as ovary. These findings suggest that Dr-DAO acts to promote ovarian maturation and that complete sexual induction depends on the production of mature ovaries. We propose that Dr-DAO produced in somatic cells prevents the onset of sexual induction in the asexual state, and then after sexual induction, the female germ cells specifically produce Dr-DAO to induce full maturation. Therefore, Dr-DAO produced in somatic and female germline cells may play different roles in sexual induction.

Structural basis of the γ-lactone-ring formation in ascorbic acid biosynthesis by the senescence marker protein-30/gluconolactonase.

The senescence marker protein-30 (SMP30), which is also called regucalcin, exhibits gluconolactonase (GNL) activity. Biochemical and biological analyses revealed that SMP30/GNL catalyzes formation of the γ-lactone-ring of L-gulonate in the ascorbic acid biosynthesis pathway. The molecular basis of the γ-lactone formation, however, remains elusive due to the lack of structural information on SMP30/GNL in complex with its substrate. Here, we report the crystal structures of mouse SMP30/GNL and its complex with xylitol, a substrate analogue, and those with 1,5-anhydro-D-glucitol and D-glucose, product analogues. Comparison of the crystal structure of mouse SMP30/GNL with other related enzymes has revealed unique characteristics of mouse SMP30/GNL. First, the substrate-binding pocket of mouse SMP30/GNL is designed to specifically recognize monosaccharide molecules. The divalent metal ion in the active site and polar residues lining the substrate-binding cavity interact with hydroxyl groups of substrate/product analogues. Second, in mouse SMP30/GNL, a lid loop covering the substrate-binding cavity seems to hamper the binding of L-gulonate in an extended (or all-trans) conformation; L-gulonate seems to bind to the active site in a folded conformation. In contrast, the substrate-binding cavities of the other related enzymes are open to the solvent and do not have a cover. This structural feature of mouse SMP30/GNL seems to facilitate the γ-lactone-ring formation.

Crystallization and preliminary crystallographic analysis of D-aspartate oxidase from porcine kidney.

D-Aspartate oxidase (DDO) from porcine kidney was crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as a precipitant. The crystal belonged to space group P2(1), with unit-cell parameters a = 79.38, b = 144.0, c = 80.46 Å, ß = 101.1°, and diffracted to 1.80 Å resolution. Molecular-replacement trials using the structure of human D-amino-acid oxidase, which is 42% identical in sequence to DDO, as a search model provided a satisfactory solution.

Rapid determination of free D-serine with chicken D-serine dehydratase.

We have developed a simple, rapid, and inexpensive method of measuring the concentration of intrinsic free D-serine in tissue samples. This method uses chicken D-serine dehydratase in an enzymatic reaction to produce pyruvate, which is detected spectrophotometrically. Pyridoxal 5'-phosphate (PLP), a cofactor of D-serine dehydratase, increased pyruvate formation by 28%. The presence of Zn(2+) or ethylenediaminetetraacetic acid (EDTA) did not have any effect on pyruvate formation under the present assay conditions. In addition, this method was not affected by the presence of a large excess of L-serine, nor by the presence of tissue extracts, and accurately determined concentrations of 2-30 µM (200 pmol-3 nmol) of D-serine. The entire assay requires only 60 min. With this method, we determined the concentration of D-serine in various silkworm tissues. The results were in agreement with high performance liquid chromatography measurements. We found high concentrations of D-serine in silkworm larvae at day 3 of the fifth instar; specifically, 509 nmol g(-1) wet tissue in the midgut, 434 nmol g(-1) in the ovary, and 353 nmol g(-1) in the testis.

Crystal structure of a zinc-dependent D-serine dehydratase from chicken kidney.

D-serine is a physiological co-agonist of the N-methyl-D-aspartate receptor. It regulates excitatory neurotransmission, which is important for higher brain functions in vertebrates. In mammalian brains, D-amino acid oxidase degrades D-serine. However, we have found recently that in chicken brains the oxidase is not expressed and instead a D-serine dehydratase degrades D-serine. The primary structure of the enzyme shows significant similarities to those of metal-activated D-threonine aldolases, which are fold-type III pyridoxal 5'-phosphate (PLP)-dependent enzymes, suggesting that it is a novel class of D-serine dehydratase. In the present study, we characterized the chicken enzyme biochemically and also by x-ray crystallography. The enzyme activity on D-serine decreased 20-fold by EDTA treatment and recovered nearly completely by the addition of Zn(2+). None of the reaction products that would be expected from side reactions of the PLP-D-serine Schiff base were detected during the >6000 catalytic cycles of dehydration, indicating high reaction specificity. We have determined the first crystal structure of the D-serine dehydratase at 1.9 Å resolution. In the active site pocket, a zinc ion that coordinates His(347) and Cys(349) is located near the PLP-Lys(45) Schiff base. A theoretical model of the enzyme-D-serine complex suggested that the hydroxyl group of D-serine directly coordinates the zinc ion, and that the ε-NH(2) group of Lys(45) is a short distance from the substrate Cα atom. The α-proton abstraction from D-serine by Lys(45) and the elimination of the hydroxyl group seem to occur with the assistance of the zinc ion, resulting in the strict reaction specificity.

Crystallization and preliminary crystallographic analysis of D-serine dehydratase from chicken kidney.

D-Serine dehydratase purified from chicken kidney was crystallized by the hanging-drop vapour-diffusion method using PEG 4000 and 2-propanol as precipitants. The crystal belonged to space group P422, with unit-cell parameters a=105.0, c=81.89 Å, and diffracted to 2.09 Šresolution. An attempt to solve the structure using the MAD method is in progress.

Immunohistochemical localization of D-aspartate oxidase in porcine peripheral tissues.

D-Aspartate (D-Asp) is an endogenous substance in mammals. Degradation of D-Asp is carried out only by D-aspartate oxidase (DDO). We measured DDO activity in porcine tissues, and produced an anti-porcine DDO antibody to examine the cellular localization of DDO. All the tissues examined showed DDO activities, whereas the substrate D-Asp was not detected in kidney cortex, liver, heart, and gastric mucosa. In the kidney, intensive immunohistochemical staining for DDO was found in the epithelial cells of the proximal tubules. In the liver, the epithelial cells of interlobular bile ducts, liver sinusoid-lining cells with cytoplasmic processes, and the smooth muscle cells of arterioles were strongly stained for DDO. In the heart, cardiomyocytes and the smooth muscle cells of arterioles showed DDO-immunoreactivity. In the gastric mucosa, only the chief cells were DDO-positive. These newly identified DDO-positive cells seem to actively degrade D-Asp to prevent an excess of D-Asp from exerting harmful effects on the respective functions of porcine tissues.

Redox control of protein conformation in flavoproteins.

Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are two flavin prosthetic groups utilized as the redox centers of various proteins. The conformations and chemical properties of these flavins can be affected by their redox states as well as by photoreactions. Thus, proteins containing flavin (flavoproteins) can function not only as redox enzymes, but also as signaling molecules by using the redox- and/or light-dependent changes of the flavin. Redox and light-dependent conformational changes of flavoproteins are critical to many biological signaling systems. In this review, we summarize the molecular mechanisms of the redox-dependent conformational changes of flavoproteins and discuss their relationship to signaling functions. The redox-dependent (or light-excited) changes of flavin and neighboring residues in proteins act as molecular "switches" that "turn on" various conformational changes in proteins, and can be classified into five types. On the basis of the present analysis, we recommend future directions in molecular structural research on flavoproteins and related proteins.

D-Serine dehydratase from chicken kidney: a vertebral homologue of the cryptic enzyme from Burkholderia cepacia.

D-serine dehydratase (DSD) catalyses the conversion of d-serine to pyruvate and ammonia. d-Serine is a physiological modulator of glutamate neurotransmission in vertebrate brains. In mammals d-serine is degraded by d-amino-acid oxidase, whereas in chicken brain it is degraded by DSD, as we have recently demonstrated [Tanaka et al. (2007) Anal. Biochem. 362, 83-88]. To clarify the roles of DSD in avian species, we purified DSD from chicken kidney. The purified enzyme was a heterodimer consisting of subunits separable by SDS-PAGE but with identical N-terminal amino acid sequences. The prominent absorption at 416 nm and the inhibition of the enzyme both by hydroxylamine and by aminooxyacetate suggested that the enzyme contains pyridoxal 5'-phosphate as a cofactor. The enzyme showed the highest specificity to d-serine: the k(cat)/K(m) values of DSD for d-serine, d-threonine and l-serine were 6.19 x 10(3), 164 and 16 M(-1)s(-1), respectively. DSD was found immunohistochemically in the proximal tubules of the chicken kidney. Judging from the amino acid sequence deduced from the cDNA, chicken DSD is a homologue of cryptic DSD from Burkholderia cepacia and low-specificity d-threonine aldolase from Arthrobacter sp. strain DK-38, all of which have a cofactor binding motif of PHXK(T/A) in their N-terminal portions.

Molecular mechanism of the redox-dependent interaction between NADH-dependent ferredoxin reductase and Rieske-type [2Fe-2S] ferredoxin.

The electron transfer system of the biphenyl dioxygenase BphA, which is derived from Acidovorax sp. (formally Pseudomonas sp.) strain KKS102, is composed of an FAD-containing NADH-ferredoxin reductase (BphA4) and a Rieske-type [2Fe-2S] ferredoxin (BphA3). Biochemical studies have suggested that the whole electron transfer process from NADH to BphA3 comprises three consecutive elementary electron-transfer reactions, in which BphA3 and BphA4 interact transiently in a redox-dependent manner. Initially, BphA4 receives two electrons from NADH. The reduced BphA4 then delivers one electron each to the [2Fe-2S] cluster of the two BphA3 molecules through redox-dependent transient interactions. The reduced BphA3 transports the electron to BphA1A2, a terminal oxygenase, to support the activation of dioxygen for biphenyl dihydroxylation. In order to elucidate the molecular mechanisms of the sequential reaction and the redox-dependent interaction between BphA3 and BphA4, we determined the crystal structures of the productive BphA3-BphA4 complex, and of free BphA3 and BphA4 in all the redox states occurring in the catalytic cycle. The crystal structures of these reaction intermediates demonstrated that each elementary electron transfer induces a series of redox-dependent conformational changes in BphA3 and BphA4, which regulate the interaction between them. In addition, the conformational changes induced by the preceding electron transfer seem to induce the next electron transfer. The interplay of electron transfer and induced conformational changes seems to be critical to the sequential electron-transfer reaction from NADH to BphA3.

Functional and structural characterization of D-aspartate oxidase from porcine kidney: non-Michaelis kinetics due to substrate activation.

D-aspartate oxidase (DDO, EC 1.4.3.1) catalyzes dehydrogenation of D-aspartate to iminoaspartate and the subsequent re-oxidation of reduced FAD with O2 to produce hydrogen peroxide. In the mammalian neuroendocrine system, D-aspartate, a natural substrate, plays important roles in the regulation of the synthesis and secretion of hormones. To elucidate the kinetic and structural properties of native DDO, we purified DDO from porcine kidney to homogeneity, cloned the cDNA, and overexpressed the enzyme in Escherichia coli. The purified DDO was a homotetramer with tightly-bound FAD. The enzyme consisted of 341 amino acids and had GAGVMG as the dinucleotide binding motif and a C-terminal SKL peroxisomal-targeting signal sequence. Porcine DDO showed a strong affinity for meso-tartrate (Kd = 118 microM). The oxidase exhibited pronounced substrate activation at D-aspartate and D-glutamate concentrations, [S], higher than 0.2 and 4 mM, respectively, and the [S]/v versus [S] plot showed marked downward curvature (v, the initial velocity), whereas substrate inhibition occurred with N-methyl-D-aspartate. These kinetic properties of DDO suggested that at high substrate concentrations, the FAD-reduced form of the enzyme also catalyzes the reaction: the oxidative half-reaction precedes the reductive one. The present direct approach to the analysis of non-Michaelis kinetics is indispensable for understanding the functional properties of DDO.