基因工程菌表达D-氨基酸氧化酶研究进展

用分子克隆手段获得D-氨基酸氧化酶基因后,对其在不同表达系统如大肠杆菌系统、酿酒酵母和克鲁维乳酸酵母、博伊丁假丝酵母、巴斯德毕赤氏酵母系统及动物细胞内的表达作了介绍。     

Resonance Raman study on the flavin in the purple intermediates of D-amino acid oxidase

Resonance Raman (RR) spectra were measured for the purple intermediates of D-amino acid oxidase reconstituted with isotopically labelled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]flavin adenine dinucleotides, and compared with those with the native enzyme. The RR lines around 1605 cm-1 with D-alanine or D-proline as a substrate and at 1548 cm-1 with D-alanine undergo isotopic shifts upon [4a-13C]- and [4,10a-13C2]-labelling. These lines are assigned to the vibrational modes associated with C(10a) = C(4a) - C(4) = O moiety of reduced flavin, providing the first assignment of RR lines of reduced flavin and conclusive evidence that reduced flavin is involved in this intermediate.     

Enhancement of the substrate specificity of D-amino acid oxidase based on tunnel-pocket engineering

D -Amino acid oxidase (DAAO) selectively catalyzes the oxidative deamination of  D -amino acids, making it one of the most promising routes for synthesizing optically pure  L -amino acids, including  L -phosphinothricin ( L -PPT), a chiral herbicide with significant market potential. However, the native DAAOs that have been reported have low activity against unnatural acid substrate  D -PPT. Herein, we designed and screened a DAAO from Rhodotorula taiwanensis (RtwDAAO), and improved its catalytic potential toward  D -PPT through protein engineering. A semirational design approach was employed to create a mutation library based on the tunnel-pocket engineering. After three rounds of iterative saturation mutagenesis, the optimal variant M_3rd-SHVG was obtained, exhibiting a >2000-fold increase in relative activity. The kinetic parameters showed that M_3rd-SHVG improved the substrate binding affinity and turnover number. This is the optimal parameter reported so far. Further, molecular dynamics simulation revealed that the M_3rd-SHVG reshapes the tunnel-pocket and corrects the direction of enzyme–substrate binding, allowing efficiently catalyze unnatural substrates. Our strategy demonstrates that the redesign of tunnel-pockets is effective in improving the activity and kinetic efficiency of DAAO, which provides a valuable reference for enzymatic catalysis. With the M_3rd-SHVG as biocatalyst, 500 mM D, L -PPT was completely converted and the yield reached 98%. The results laid the foundation for further industrial production.     

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.     

Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20–60°C) and protein concentration (5–100 µmol L^?1). The time courses of loss of enzyme activity in 100 mmol L^?1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k_a) increased 10⁴-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (k_b) and binding (k_?b) as well as the irreversible aggregation of the apo-enzyme (k_agg) were less sensitive to changes in temperature, their activation energy (E_a) being about 52 kJ mol^?1 in comparison with an E_a value of 185 kJ mol^?1 for k_a. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5′-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L^?1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.     

Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20-60°C) and protein concentration (5-100 µmol L^-1). The time courses of loss of enzyme activity in 100 mmol L^-1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k _a) increased 10⁴-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (k _b) and binding (k _-b) as well as the irreversible aggregation of the apo-enzyme (k _agg) were less sensitive to changes in temperature, their activation energy (E _a) being about 52 kJ mol^-1 in comparison with an E _a value of 185 kJ mol^-1 for k _a. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5'-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L^-1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.     

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。     

Coimmobilization of D-amino acid oxidase and catalase by entrapment ofTrigonopsis variabilis in radiation polymerised Polyacrylamide beads

Trigonopsis variabilis induced for D-amino acid oxidase and catalase was immobilized by entrapment in Polyacrylamide beads obtained by radiation polymerisation. Permeabilization of the cells was found to be essential for optimal activity of the enzymes in free cells. However, the process of entrapment itself was found to eliminate the permeability barrier of cells immobilized in Polyacrylamide. The two enzymes exhibited a differential response on Polyacrylamide entrapment. Thus, D-amino acid oxidase activity was stabilized to heat inactivation whereas catalase in the same cells showed a destabilization on entrapment in Polyacrylamide. The coimmobilized enzyme preparation showed an operational half life of 7–9 days after which the D-amino acid oxidase activity remained stable at a value 35–40% of that of the initial activity for a study period of 3 weeks. Coimmobilization of MnO₂ was not effective in enhancing the operational life of the enzyme preparation.     

Genetic loss of D-amino acid oxidase activity reverses schizophrenia-like phenotypes in mice

Reduced function of the N -methyl- d -aspartate receptor (NMDAR) has been implicated in the pathophysiology of schizophrenia. The NMDAR contains a glycine binding site in its NR1 subunit that may be a useful target for the treatment of schizophrenia. In this study, we assessed the therapeutic potential of long-term increases in the brain levels of the endogenous NMDAR glycine site agonist D-serine, through the genetic inactivation of its catabolic enzyme D-amino acid oxidase (DAO) in mice. The effects of eliminating DAO function were investigated in mice that display schizophrenia-related behavioral deficits due to a mutation ( Grin 1 ^D481N ) in the NR1 subunit that results in a reduction in NMDAR glycine affinity. Grin 1 ^D481N mice show deficits in sociability, prolonged latent inhibition, enhanced startle reactivity and impaired spatial memory. The hypofunctional Dao 1 ^G181R mutation elevated brain levels of D-serine, but alone it did not affect performance in the behavioral measures. Compared to animals with only the Grin 1 ^D481N mutation, mice with both the Dao1 ^G181R and Grin 1 ^D481N mutations displayed an improvement in social approach and spatial memory retention, as well as a reversal of abnormally persistent latent inhibition and a partial normalization of startle responses. Thus, an increased level of D-serine resulting from decreased catalysis corrected the performance of mice with deficient NMDAR glycine site activation in behavioral tasks relevant to the negative and cognitive symptoms of schizophrenia. Diminished DAO activity and elevations in D-serine may serve as an effective therapeutic intervention for the treatment of psychiatric symptoms.     

D-Amino acid oxidase: Physiological role and applications

D-Amino acids play a key role in regulation of many processes in living cells. FAD-dependent D-amino acid oxidase (DAAO) is one of the most important enzymes responsible for maintenance proper level of D-amino acids. The most interesting and important data for regulation of the nervous system, hormone secretion, and other processes by D-amino acids as well as development of different diseases under changed DAAO activity are presented. The mechanism of regulation is complex and multi-parametric because the same enzyme simultaneously influences the level of different D-amino acids, which can result in opposing effects. Use of DAAO for diagnostic and therapeutic purposes is also considered.     

Optimization of culture condition for the production of D-amino acid oxidase in a recombinant Escherichia coli

The gene encoding D-amino acid oxidase (DAAO) from Trigonopsis variabilis CBS 4095 has been cloned and expressed in Escherichia coli BL21 (DE3). Unfortunately, it was observed that the host cell was negatively affected by the expressed DAAO, resulting in a remarkable decrease in cell growth. To overcome this problem, we investigated several factors that affect cell growth rate and DAAO production such as addition time of inducer and dissolved oxygen (DO) concentration. The addition time of lactose, which was used as an inducer, and DO concentration appeared to be critical for the cell growth of E. coli BL21 (DE3)/pET-DAAO. A two-stage DO control strategy was developed, in which the DO concentration was controlled above 50% until specific stage of bacterial growth (OD₆₀₀ 30–40) and then downshifted to 30% by changing the agitation speed and aeration rate, and they remained at these rates until the end of fermentation. With this strategy, the maximum DAAO activity and cell growth reached 18.5 U/mL and OD₆₀₀ 81, respectively. By reproducing these optimized conditions in a 12-m³ fermentor, we were able to produce DAAO at a productivity of 19 U/mL with a cell growth of OD₆₀₀ 80.     

D-amino acid oxidase: structure,catalytic mechanism,and practical application

D-Amino acid oxidase (DAAO) is a FAD-dependent enzyme that plays an important role in microbial metabolism, utilization of endogenous D-amino acids, regulation of the nervous system, and aging in mammals. DAAO from yeasts Rhodotorula gracilis and Trigonopsis variabilis are used to convert cephalosporin C into 7-aminocephalosporanic acid, the precursor of other semi-synthetic cephalosporins. This review summarizes the recent data on the enzyme localization, physiological role, gene cloning and expression, and the studies on the enzyme structure, stability, catalytic mechanism, and practical applications.Translated from Biokhimiya, Vol. 70, No. 1, 2005, pp. 51–67.Original Russian Text Copyright © 2005 by Tishkov, Khoronenkova.     

Characterization of human D-amino acid oxidase

D-Amino acid oxidase (DAAO) has been proposed to be involved in the oxidation of D-serine, an allosteric activator of the NMDA-type glutamate receptor in the brain, and to be associated with the onset of schizophrenia. The recombinant human DAAO was expressed in Escherichia coli and was isolated as an active homodimeric flavoenzyme. It shows the properties of the dehydrogenase-oxidase class of flavoproteins, possesses a low kinetic efficiency, and follows a ternary complex (sequential) kinetic mechanism. In contrast to the other known DAAOs, the human enzyme is a stable homodimer even in the apoprotein form and weakly binds the cofactor in the free form.     

Production of D-amino acid oxidase (DAO) of Trigonopsis variabilis in Schizosaccharomyces pombe and the characterization of biocatalysts prepared with recombinant cells

The cDNA of D-amino acid oxidase (DAO) gene isolated from Trigonopsis variabilis was expressed in Schizosaccharomyces pombe. A clone, ASP327-10, transformed with plasmid vector, pTL2M5DAO, expressed catalytically active DAO in the presence of G418, and converted Cephalosprin C to alpha-ketoadipyl-7-cephalosporanic acid (KA-7-ACA) and glutaryl-7-aminocephalosporanic acid (GL-7-ACA). Biocatalysts were prepared using ASP327-10 and T. variabilis, and evaluated to demonstrate the feasibility of recombinant S. pombe for industrial application. The cells were immobilized by crosslinking polyethylene imine after glutardialdehyde (GDA) fixation and permeabilization by alkaline treatment. Although the biocatalyst prepared from ASP327-10 exhibited DAO activity, catalase activity still remained fully even after permeabilization, under which condition, the catalase activity of T. variabilis decreased to 20-30%. Heat treatment was required before cell fixation by GDA to inactivate the catalase in S. pombe. This improved the efficiency of bioconversion to GL-7-ACA, but caused poor mechanical strength in the biocatalyst of S. pombe. To overcome this weakness, a catalase-deficient host strain was obtained by ethylmethansulfate mutagenesis. Moreover, taking economics into consideration, the integrative vector, pTL2M5DAO-8XL, with multi-copies of expression cassette was constructed to express DAO in S. pombe even in the absence of G418. The newly established integrant, ASP417-7, did not exhibit any catalase activity so that heat treatment was not required. The obtained integrant and its biocatalyst were significantly improved in GL-7ACA conversion ability and mechanical strength. This study demonstrates that the established integrant is a potential candidate as an alternative source of DAO enzyme.     

三角酵母D-氨基酸氧化酶基因在大肠杆菌中的克隆和表达

从三角酵母中提取总RNA ,反转录后进行PCR扩增得到D 氨基酸氧化酶 (D AminoAcidOxidase ,DAAO)基因 ,经测序可知 ,与文献中三角酵母的DAAO基因序列的同源性在 99%以上。将DAAO基因用NcoⅠ和BamHⅠ双酶切后 ,与相同酶切的大肠杆菌表达载体pET 2 8a连接 ,转化大肠杆菌TOP 1 0F′,并筛选得到重组质粒pET DAAO ,转化BL2 1 (DE3)感受态细胞 ,得到重组大肠杆菌BL2 1 (DE3) pET DAAO。对重组大肠杆菌中的D 氨基酸氧化酶进行了诱导表达 ,考察了诱导温度、菌浓度、诱导剂IPTG用量以及溶氧等因素对酶活的影响。结果表明 ,在 2 8℃、菌浓度 (OD6 0 0 ) 1 0、IPTG浓度 1mmol L时 ,DAAO酶活最高达 2 3 3U mL。研究进一步显示 ,用廉价无毒的乳糖可以替代IPTG进行诱导 ,当乳糖浓度为 2mmol L ,DAAO酶活可达 2 2 7U mL。经过补料分批培养和乳糖诱导 ,DAAO酶活可以达到 1 75U mL。     

A novel neuropeptide precursor gene is expressed in the terrestrial snail central nervous system by a group of neurons that control mating behavior

We report the isolation of a cDNA clone encoding a neuropeptide precursor named preproGFAD from the central nervous system (CNS) of the snail Helix lucorum. Analysis of the expression of this gene shows that it is neurospecific and expressed in several groups of CNS neurons. Most notable is the expression of preproGFAD gene in the right mesocerebrum, where the neurons controlling mating behavior are located. The expression in this particular region is observed in adult animals but not in juvenile ones. The preprohormone is 108 amino acids long and contains a hydrophobic leader peptide and eight Lys-Arg recognition sites for endoproteolysis. The post-translational processing of the prohormone may lead to the generation of seven tetrapeptides, Gly-Phe-Ala-Asp-COOH (GFAD). This peptide has the same sequence as two previously isolated peptides from a related snail, Achatina fulica. The first of them (achatin-I) contains D-Phe; the second (achatin-II) is its L-Phe-containing stereoisomer. Injection of synthetic D-GFAD in nanomolar concentrations into intact animals caused an increase of the heartbeat rate and opening of the genital atrium. In preparations containing CNS with intact innervation of reproductive organs, bath application of D-GFAD caused extensive movements of the penis but not of other reproductive organs. Intracellular activation of individual neurons expressing the preproGFAD gene also elicited penis movements. D-GFAD also suppressed activity of neurons modulating feeding behavior. Our data therefore indicate that the preproGFAD gene encodes the precursor of a neuropeptide that participates in the regulation of male mating behavior. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 183–197, 1998     

Crystal structure of human D-amino acid oxidase: context-dependent variability of the backbone conformation of the VAAGL hydrophobic stretch located at the si-face of the flavin ring

In the brain, the extensively studied FAD-dependent enzyme D-amino acid oxidase (DAO) degrades the gliotransmitter D-serine, a potent activator of N-methyl-D-aspartate type glutamate receptors, and evidence suggests that DAO, together with its activator G72 protein, may play a key role in the pathophysiology of schizophrenia. Indeed, its potential clinical importance highlights the need for structural and functional analyses of human DAO. We recently succeeded in purifying human DAO, and found that it weakly binds FAD and shows a significant slower rate of flavin reduction compared with porcine DAO. However, the molecular basis for the different kinetic features remains unclear because the active site of human DAO was considered to be virtually identical to that of porcine DAO, as would be expected from the 85% sequence identity. To address this issue, we determined the crystal structure of human DAO in complex with a competitive inhibitor benzoate, at a resolution of 2.5 Angstrom. The overall dimeric structure of human DAO is similar to porcine DAO, and the catalytic residues are fully conserved at the re-face of the flavin ring. However, at the si-face of the flavin ring, despite the strict sequence identity, a hydrophobic stretch (residues 47-51, VAAGL) exists in a significantly different conformation compared with both of the independently determined porcine DAO-benzoate structures. This suggests that a context-dependent conformational variability of the hydrophobic stretch accounts for the low affinity for FAD as well as the slower rate of flavin reduction, thus highlighting the unique features of the human enzyme.     

Properties and chemical modification of D-amino acid oxidase from Trigonopsis variabilis

The basic properties of purified d-amino acid oxidase from the yeast Trigonopsis variabilis were investigated. The pH optimum of activity was between pH 8.5 and 9.0, and the native molecular masses of holo- and apo-enzyme were determined to be 170 kDa; higher aggregates corresponded to molecular masses of 320 and 570 kDa. The apparent V _max and K _m values for different substrates varied between 3.7 to 185 U/mg and 0.2 to 17.3 mM, respectively. The reaction of d-amino acid oxidase with sulfite was followed by the typical spectral modifications of the FAD resembling the reduced enzyme; a K _d of 30 μM was calculated for the N(5)-adduct. The red anionic flavin radical of the enzyme was stable; benzoate had no influence on the spectral properties. A complete loss of enzyme activity was observed after chemical modification by the histidine-specific reagent diethyl pyrocarbonate. The inactivation showed pseudo-first-order kinetics, with a second-order rate constant of 13.6 M^–1 min^–1 at pH 6.0 and 20°C. The addition of a substrate under anoxic conditions led to a substantial protection from inactivation, which indicates a localization of the modified residues close to the active site. The pK_a of the reacting group was determined to be 7.7, and the rate of inactivation reached a limiting value of 0.031 min^–1. Received: 22 August 1995 / Accepted: 17 October 1995     

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.