D-氨基酸氧化酶研究进展

D-氨基酸氧化酶(D-amino acid oxidase:oxidoreductase, DAAO, EC 1.4.3.3)是一种以黄素腺嘌呤(FAD)为辅基的典型黄素蛋白酶类,可氧化D-氨基酸的氨基生成相应的酮酸和氨。在体内D-氨基酸的代谢中起着重要作用。主要介绍了D-氨基酸氧化酶的生理功能和应用、表达条件优化及通过定点突变对酶学性质的研究。     

D-aspartate oxidation by rat and bovine renal peroxisomes: an electron microscopic cytochemical study

D-amino acid oxidase, a peroxisomal enzyme, and D-aspartate oxidase, a potential peroxisomal enzyme, share biochemical attributes. Both produce hydrogen peroxide in flavin-requiring oxidative reactions. Such similarities suggest that D-aspartate oxidase may also be localized to peroxisomes. Definitive identification of D-aspartate oxidase as a peroxisomal enzyme depends, however, on visualization at the electron microscopic level. Using incubation conditions shown to be specific for the enzyme in biochemical studies, this report extends the cytochemical localization of D-amino acid oxidase to bovine renal peroxisomes, and shows that D-aspartate can be oxidized by rat and bovine renal peroxisomes. An unexpected finding was the sensitivity of both D-amino acid oxidase activity (proline specific) and D-aspartate oxidase activity to inhibition by agents used in biochemical studies to discriminate between the two enzyme activities. Therefore, it is possible that, in the cytochemical system used in this study, (a) either D-proline and D-aspartate are substrates for only one enzyme or (b) the two enzymes have additional overlapping biochemical properties.     

Thermodynamic control of D-amino acid oxidase by benzoate binding

The redox properties of D-amino acid oxidase (D-amino-acid: O2 oxidoreductase (deaminating) EC1.4.3.3) have been measured at 18 degrees C in 20 mM sodium pyrophosphate, pH 8.5, and in 50 mM sodium phosphate, pH 7.0. Over the entire pH range, 2 eq are required per mol of FAD in D-amino acid oxidase for reduction to the anion dihydroquinone. The red anion semiquinone is thermodynamically stable as indicated by the separation of the electron potentials and the quantitative formation of the semiquinone species. The first electron potential is pH-independent at -0.098 +/- 0.004 V versus SHE while the second electron potential is pH-dependent exhibiting a 0.060 mV/pH unit slope. The redox behavior of D-amino acid oxidase is consistent with that observed for other oxidase enzymes. On the other hand, the behavior of the benzoate-bound enzyme under the same conditions is in marked contrast to the thermodynamics of free D-amino acid oxidase. Spectroelectrochemical experiments performed on inhibitor-bound (benzoate) D-amino acid oxidase show that benzoate binding regulates the redox properties of the enzyme, causing the energy levels of the benzoate-bound enzyme to be consistent with the two-electron transfer catalytic function of the enzyme. Our data are consistent with benzoate binding at the enzyme active site destroying the inductive effect of the positively charged arginine residue. Others have postulated that this positively charged group near the N(1)C(2) = O position of the flavin controls the enzyme properties. The data presented here are the clearest examples yet of enzyme regulation by substrate which may be a general characteristic of all flavoprotein oxidases.     

Reactive oxygen species (ROS)-generating oxidases in the normal rabbit cornea and their involvement in the corneal damage evoked by UVB rays

The corneas of albino rabbits were irradiated (5 min exposure once a day) with UVB rays (312 nm) for 4 days (shorter procedure) or 8 days (longer procedure). The eyes were examined microbiologically and only the corneas of sterile eyes or eyes with non-pathogenic microbes were employed. Histochemically, the activities of reactive oxygen species (ROS)-generating oxidases (xanthine oxidase, D-amino acid oxidase and alpha-hydroxy acid oxidase) were examined in cryostat sections of the whole corneas. Biochemically, the activity of xanthine oxidoreductase/xanthine oxidase was investigated in the scraped corneal epithelium. UVB rays significantly changed enzyme activities in the corneas. In comparison to the normal cornea, where of ROS-generating oxidases only xanthine oxidase showed significant activity in the corneal epithelium and endothelium, D-amino acid oxidase was very low and alpha-hydroxy acid oxidase could not be detected at all, in the cornea repeatedly irradiated with UVB rays, increased activities of xanthine oxidase and D-amino acid oxidase were observed in all corneal layers. Only after the longer procedure the xanthine oxidase and D-amino acid oxidase activities were decreased in the thinned epithelium in parallel with its morphological disturbances. Further results show that the xanthine oxidase/xanthine oxidoreductase ratio increased in the epithelium together with the repeated irradiation with UVB rays. This might suggest that xanthine dehydrogenase is converted to xanthine oxidase. However, in comparison to the normal corneal epithelium, the total amount of xanthine oxidoredutase was decreased in the irradiated epithelium. It is presumed that xanthine oxidoreductase might be released extracellularly (into tears) or the enzyme molecules were denatured due to UVB rays (particulary after the longer procedure). Comparative histochemical and biochemical findings suggest that reactive oxygen species-generating oxidases (xanthine oxidase, D-amino acid oxidase) contribute to the corneal damage evoked by UVB rays.     

透明颤菌血红蛋白对两种D-氨基酸氧化酶的不同影响及机理研究

D-氨基酸氧化酶是两步酶法制备7-氨基头孢烷酸(7-ACA)这一半合成头孢类抗生素的主要前体的关键酶.它催化的反应是需氧反应,反应体系的溶氧水平是酶活的限制因素之一.我们发现将纯化的透明颤菌血红蛋白(VHb)分别添加到三角酵母来源(TvDAO)和红酵母来源(RgDAO)的D-氨基酸氧化酶的纯酶中,可提高这两种氧化酶的活力35%和48%.细菌双杂交实验证明,透明颤菌血红蛋白与RgDAO有相互作用,而与TvDAO没有关联.这说明透明颤菌血红蛋白对氧化酶活力的促进是由于自身向氧化酶提供游离氧,而且它与氧化酶之间的相互作用可以增强这种效果.我们可以利用透明颤菌血红蛋白的这种性质把它作为氧化酶酶促反应的添加剂,提高酶促反应的效率,如果该氧化酶与之有相互作用,效果会更加显著.     

荧光假单胞菌TM5-2中D-型氨基酸脱氢酶的鉴定和表达

从荧光假单胞菌TM5-2中得到一个含丙氨酸消旋酶基因的DNA片段(8.8kb),相邻的一个开读框(ORF)与甘氨酸/D-型氨基酸氧化酶基因相似。该ORF经过克隆、表达,并没有检测到甘氨酸/D-型氨基酸氧化酶的活性,推导而得的氨基酸序列与D-型氨基酸脱氢酶序列比较发现,ORF含有D-型氨基酸脱氢酶的所有重要的保守序列。经TTC培养基鉴定,其具有D-型氨基酸脱氢酶的活性,并对一系列D-型氨基酸有作用,最佳作用底物是D-组氨酸。     

Catalytic and structural characteristics of carp hepatopancreas D-amino acid oxidase expressed in Escherichia coli

D-amino acid oxidase of carp (Cyprinus carpio) hepatopancreas was overexpressed in Escherichia coli cells and purified to homogeneity for the first time in animal tissues other than pig kidney. The purified preparation had a specific activity of 293 units mg(-1) protein toward D-alanine as a substrate. It showed the highest activity toward D-alanine with a low Km of 0.23 mM and a high kcat of 190 s(-1) compared to 10 s(-1) of the pig kidney enzyme. Nonpolar and polar uncharged D-amino acids were preferable substrates to negatively or positively charged amino acids. The enzyme exhibited better thermal and pH stabilities than several yeast counterparts or the pig kidney enzyme. Secondary structure topology consisted of 11 alpha-helices and 17 beta-strands that differed slightly from pig kidney and Rhodotorula gracilis enzymes. A three-dimensional model of the carp enzyme constructed from a deduced amino acid sequence resembled that of pig kidney D-amino acid oxidase but with a shorter active site loop and a longer C-terminal loop. Judging from these characteristics, carp D-amino acid oxidase is close to the pig kidney enzyme structurally, but analogous to the R. gracilis enzyme enzymatically in turnover rate and pH and temperature stabilities.     

A D-amino acid oxidase from Chlorella vulgaris.

A procedure has been developed for the partial purification from Chlorella vulgaris of an enzyme which catalyzes the formation of HCN from D-histidine when supplemented with peroxidase of a metal with redox properties. Some properties of the enzyme are described. Evidence is presented that the catalytic activity for HCN formation is associated with a capacity for catalyzing the oxidation of a wide variety of D-amino acids. With D-leucine, the best substrate for O2 consumption, 1 mol of ammonia is formed for half a mol of O2 consumed in the presence of catalase. An inactive apoenzyme can be obtained by acid ammonium sulfate precipitation, and reactivated by added FAD. On the basis of these criteria, the Chlorella enzyme can be classified as a D-amino acid oxidase (EC 1.4.3.3). Kidney D-amino acid oxidase and snake venom L-amino acid oxidase, which likewise form HCN from histidine on supplementation with peroxidase, have been compared with the Chlorella D-amino acid oxidase. The capacity of these enzymes for causing HCN formation from histidine is about proportional to their ability to catalyze the oxidation of histidine.     

Translation of messenger RNA of pig kidney D-amino acid oxidase in a cell-free system

In vitro synthesis of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3], one of the peroxisomal flavin enzymes, was performed using a rabbit reticulocyte lysate system in order to elucidate the biosynthetic pathway of the enzyme. The apparent molecular weight of the synthesized enzyme protein was the same as that of D-amino acid oxidase purified from pig kidney. On the other hand, the enzyme protein was not detectable when a wheat germ lysate system was used for the translation. Denaturation of pig kidney poly(A)+ RNA with methylmercury hydroxide prior to the translation was found to enhance the synthesis of the enzyme protein. These results suggest a tight conformational structure of the mRNA used.     

Exchange of free and bound coenzyme of flavin enzymes studied with [14C]FAD.

The exchange of bound FAD for free FAD was studied with D-amino acid oxidase (D-amino acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3) and beta-D-glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4). For a simple measurement of the reaction rate, equimolar amounts of the enzyme and [14C]FAD were mixed. The exchange occurred very rapidly in the holoenzyme of D-amino acid oxidase at 25 degrees C, pH 8.3 (half life of the exchange: 0.8 min), but slowly in the presence of the substrate or a competitive inhibitor, benzoate. It also occurred slowly in the purple complex of D-amino acid oxidase. In the case of beta-D-glucose oxidase, however, the exchange occurred very slowly at 25 degrees C, pH 5.6, regardless of the presence of the substrate or p-chloromercuribenzoate. On the basis of these findings, the turnover of the coenzymes of flavin enzymes in mammals is discussed.     

Thermostabilization of porcine kidney D-amino acid oxidase by a single amino acid substitution

D-amino acid oxidase (DAO) is of considerable practical importance, such as bioconversion and enzymatic assay. In this study, we succeeded in obtaining a thermostable mutant DAO from porcine kidney by a single amino acid substitution. This mutant enzyme, F42C, was stable at 55 degrees C, while the wild-type enzyme was stable only up to 45 degrees C. The Km values of F42C for D-amino acids was about half of those of the wild-type enzyme. This mutant DAO with improved stability and affinity for its substrates is advantageous for the determination of D-amino acids.     

Effect of hydrogen peroxide on d-amino acid oxidase from Rhodotorula gracilis

D-amino acid oxidase from Rhodotorula gracilis is a FAD-containing enzyme that belongs to the oxidase class that is characterized by the ability of the reduced flavin to react quickly with oxygen, yielding hydrogen peroxide and the oxidized cofactor. Hydrogen peroxide, necessary for the production of glutaryl-7-ACA from cephalosporin C had a deleterious effect on the enzyme. H(2)O(2) induced the oxidation of tryptophan and cysteine residues of the protein that could be involved in the dimerization process, required for the attainment of a fully competent enzyme. H(2)O(2) had also a kinetic effect on the reaction catalyzed by D-amino acid oxidase. It was a pure noncompetitive inhibitor; the corresponding inhibition constants were K(is) = 0.52 mM and K(ii) = 0.70 mM.     

Isolation and characterization of a mitochondrial D-amino acid oxidase from Neurospora crassa.

D-Amino acid oxidase (EC 1.4.3.3) activity in homogenates of Neurospora crassa strain SY7A was found to sediment with the mitochondrial fraction. Digitonin fractionation studies on purified mitochondria have indicated a matrix localization of the enzyme. Additionally, a peroxidase (EC 1.11.1.7) activity, which may remove hydrogen peroxide formed as a product of D-amino acid oxidation, was also found in the mitochondrial matrix. Partial purification (20- to 30-fold) of the mitochondrial D-amino acid oxidase was achieved. The enzyme exhibited a pH optimum between 9.0 and 9.2, temperature optimum between 20 and 30 degrees C, and a molecular weight of 118 000 +/- 6000 as determined by gel electrophoresis and 125 000 as determined by gel chromatography.     

The primary structure of D-amino acid oxidase from Rhodotorula gracilis

Summary The amino acid sequence of D-amino acid oxidase from Rhodotorula gracilis was determined by automated Edman degradation of peptides generated by enzymatic and chemical cleavage. The enzyme monomer contains 368 amino acid residues and its sequence is homologous to that of other known D-amino acid oxidases. Six highly conserved regions appear to have a specific role in binding of coenzyme FAD, in active site topology and in peroxisomal targeting. Moreover, Rhodotorula gracilis D-amino acid oxidase contains a region with a cluster of basic amino acids, probably exposed to solvent, which is absent in other D-amino acid oxidases.     

Temperature-induced changes in the coenzyme environment of D-amino acid oxidase revealed by the multiple decays of FAD fluorescence.

A temperature-dependent change in the microenvironment of the coenzyme, FAD, of D-amino acid oxidase was investigated by means of steady-state and picosecond time-resolved fluorescence spectroscopy. Relative emission quantum yields from FAD bound to D-amino acid oxidase revealed the temperature transition when concentration of the enzyme was lowered. The observed fluorescence decay curves were well described with four-exponential decay functions. The amplitude of the shortest lifetime (tau 0), approximately 25 ps, was always negative, which indicates that the fluorescence of D-amino acid oxidase at approximately 520 nm appears after a metastable state of the excited isoalloxazine decays. The other components with positive amplitudes were assigned to dimer or associated forms of the enzyme, monomer, and free FAD dissociated from the enzyme. Ethalpy and entropy changes of intermediate states in the quenching processes were evaluated according to the absolute rate theory. The temperature transition was much more pronounced in the monomer than in the dimer or associated forms of the enzyme.     

Theoretical approaches to D-amino acid oxidase

Several substrates and roles have been proposed for D-amino acid oxidase (E.C. 1.4.3.3.); however, there is no proof that they possess the required characteristics to account for the ubiquity, large amounts and great activity of the enzyme as found in diverse cells and tissues. Based on the similar stereoposition of identically charged atoms and lateral side chain (R) with respect to the alpha-hydrogen atoms in beta-sheet conformation and in D-amino acids, it is proposed that its substrates may include several membrane-related proteins, partially in beta-sheet conformation, whose alpha-hydrogen atoms would be the real object of D-amino acid oxidase catalysis. A monooxygenase-like enzymatic activity of D-amino acid oxidase with these novel substrates is considered, for which the final products are hypothesized to be protein alpha-carbon hydroxyls resulting from the incorporation of one atom of oxygen into the substrate, the other being reduced to water. Alternatively, it is also proposed that D-amino acid oxidase (and possibly other monooxygenase enzymes) would have a hydroperoxide-synthetase activity. In this case, protein alpha-carbon hydroperoxide and not water, but another reduced molecule, would be the final products. The new enzymatic performances of D-amino acid oxidase and the possible role of its potential final products in redox and other biochemical processes are discussed.     

Expression of D-amino acid oxidase in Rhodotorula gracilis under induction conditions: a biochemical and cytochemical study.

D-amino acid oxidase is expressed to a high level in the yeast Rhodotorula gracilis (0.3% of total cell protein) through induction by D-alanine in a defined growth medium. Monospecific polyclonal antibodies against pure enzyme were obtained. Western blot analysis showed that the enzyme is synthesized as the mature polypeptide. The localization of the enzyme was investigated by immunoelectron microscopy using the postembedding immunogold technique and by submicroscopic enzyme cytochemistry. D-Amino acid oxidase was detected in peroxisomes, and quantitation of immunoelectron microscopic data indicated that the enzyme is exclusively confined to these organelles. Immunoelectron microscopic observations are in complete agreement with biochemical data showing that the enzyme is not expressed in the absence of D-alanine. Morphometric analysis demonstrated that induction of D-amino acid oxidase synthesis is associated with a 241% increase of peroxisome volume density and with a 31% increase of peroxisome size as compared to cells grown on non-inducing medium.     

Evaluation of D-amino acid oxidase from Rhodotorula gracilis for the production of alpha-keto acids: a reactor system

The study reports on the development of a bioreactor for the production of alpha-keto acids from D,L- or D-amino acids using Rhodotorula gracilis D-amino acid oxidase. D-Amino acid oxidase was co-immobilized with catalase on Affi-Gel 10 matrix, and the reactor was operated as a continuous-stirred tank reactor (CSTR) or stirred tank with medium recycling conditions. The optimum substrate concentration and quantity of biocatalyst were determined (5 mM and 1.2 mg/L, respectively). Under optimum operating conditions, product formation was linearly related to both substrate and enzyme concentration, showing the system to be highly flexible. Under these conditions, in a stirred tank, over 90% conversion was achieved in 30 min with a maximum production of 0.23 g of pyruvic acid/day/enzyme units. Product was recovered by ion exchange chromatography. The operational stability of the reactor was high (up to 9.5 h of operation without loss of activity) and the inactivation half-life was not reached even after 18 h or 36 bioconversion cycles. This represents the first case of a reactor developed successfully with a D-amino acid oxidase. (c) 1994 John Wiley & Sons, Inc.     

Immunoelectron microscopic study of a new D-amino acid oxidase-immunoreactive subcompartment in rat liver peroxisomes.

We report the presence of a new subcompartment in rat liver peroxisomal matrix in which only D-amino acid oxidase is localized and other matrix enzymes are absent. By electron microscopic observation, the rat liver peroxisome has generally been considered to consist of a single limiting membrane, an electron-dense crystalline core, and a homogeneous matrix. Immunohistochemical staining for D-amino acid oxidase by the protein A-gold technique revealed the presence of a small area in the matrix that was immunoreactive for the enzyme and was less electron-dense than the surrounding matrix. The localization of D-amino acid oxidase in this small area of the peroxisomal matrix was confirmed by immunoelectron microscopy on freeze-substituted tissues processed without chemical fixation. To analyze the characteristics of the electron-lucent area, immunoreactivity for various peroxisomal enzymes, including catalase, acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, 3-ketoacyl-CoA thiolase, L-alpha-hydroxy acid oxidase (isozyme B), and glycolate oxidase (isozyme A), was assayed. The electron-lucent area was negative for all of these. By double staining for D-amino acid oxidase and catalase, using colloidal gold particles of different sizes, these enzymes were shown to be located in separate areas in the matrix.