Expression Of D-amino Acid Oxidase Research Articles

Spinal d-Amino Acid Oxidase Contributes to Neuropathic Pain in Rats

D-amino acid oxidase (DAO) is an enzyme catalyzing oxidative deamination of neutral and polar d-amino acids and is expressed in the kidneys, liver, and central nervous system (CNS) including the spinal cord. We have previously demonstrated that DAO gene deletion/mutation by using mutant ddY/DAO(-/-) mice and systemic administration of the DAO inhibitor sodium benzoate blocked formalin-induced hyperalgesia in mice. In this study, we further investigated the potential role of DAO in neuropathic pain in a rat model of tight L(5)/L(6) spinal nerve ligation. After L(5)/L(6) spinal nerve ligation, the mRNA expression (measured by real-time quantitative polymerase chain reaction) and enzyme activity (measured by a colorimetric method) of DAO in the lumbar spinal cord were markedly increased, in agreement with the development of neuropathic pain (mechanical allodynia). Intraperitoneal injection of sodium benzoate (400 mg/kg) specifically blocked mechanical allodynia in neuropathic rats and formalin-induced hyperalgesia but did not suppress acute pain responses in the tail-flick test or formalin test. Systemic injection of sodium benzoate also inhibited DAO activity in the lumbar spinal cord of rats. Furthermore, direct intrathecal (spinal cord) injection of benzoate (30 mug/rat) specifically blocked spinal nerve ligation-induced mechanical allodynia in neuropathic rats and formalin-induced hyperalgesia (but not acute pain) in the formalin test. Based on the above results, we conclude that spinal DAO plays a pronociceptive (rather than an antinociceptive) role and might be a target molecule for the treatment of chronic pain of neuropathic origin.

Increased d-amino acid oxidase expression in the bilateral hippocampal CA4 of schizophrenic patients: a post-mortem study

An important risk gene in schizophrenia is d-amino acid oxidase (DAAO). To establish if expression of DAAO is altered in cortical, hippocampal or thalamic regions of schizophrenia patients, we measured gene expression of DAAO in a post-mortem study of elderly patients with schizophrenia and non-affected controls in both hemispheres differentiating between gray and white matter. We compared cerebral post-mortem samples (granular frontal cortex BA9, middle frontal cortex BA46, superior temporal cortex BA22, entorhinal cortex BA28, sensoric cortex BA1–3, hippocampus (CA4), mediodorsal nucleus of the thalamus) from 10 schizophrenia patients to 13 normal subjects investigating gene expression of DAAO in the gray and white matter of both hemispheres of the above-mentioned brain regions by in situ-hybridization. We found increased expression of DAAO-mRNA in the hippocampal CA4 of schizophrenic patients. Compared to the control group, both hemispheres of the hippocampus of schizophrenic patients showed an increased expression of 46% (right, P = 0.013) and 54% (left, P = 0.019), respectively. None of the other regions examined showed statistically significant differences in DAAO expression. This post-mortem study demonstrated increased gene expression of DAAO in the left and right hippocampus of schizophrenia patients. This increased expression could be responsible for a decrease in local d-serine levels leading to a NMDA-receptor hypofunction that is hypothesized to play a major role in the pathophysiology of schizophrenia. However, our study group was small and results should be verified using larger samples.

Reduced serine racemase expression contributes to age-related deficits in hippocampal cognitive function

To gain insight into the contribution of d-serine to impaired cognitive aging, we compared the metabolic pathway and content of the amino acid as well as d-serine-dependent synaptic transmission and plasticity in the hippocampus of young and old rats of the Wistar and Lou/C/Jall strains. Wistar rats display cognitive impairments with aging that are not found in the latter strain, which is therefore considered a model of healthy aging. Both mRNA and protein levels of serine racemase, the d-serine synthesizing enzyme, were decreased in the hippocampus but not in the cerebral cortex or cerebellum of aged Wistar rats, whereas the expression of d-amino acid oxidase, which degrades the amino acid, was not affected. Consequently, hippocampal levels of endogenous d-serine were significantly lower. In contrast, serine racemase expression and d-serine levels were not altered in the hippocampus of aged Lou/C/Jall rats. Ex vivo electrophysiological recordings in hippocampal slices showed a marked reduction in N-methyl-d-aspartate-receptor (NMDA-R)-mediated synaptic potentials and theta-burst-induced long-term potentiation (LTP) in the CA1 area of aged Wistar rats, which were restored by exogenous d-serine. In contrast, NMDA-R activation, LTP induction and responses to d-serine were not altered in aged Lou/C/Jall rats. These results further strengthen the notion that the serine racemase-dependent pathway is a prime target of hippocampus-dependent cognitive deficits with aging. Understanding the processes that specifically affect serine racemase during aging could thus provide key insights into the treatment of memory deficits in the elderly.

The neurobiology of D-amino acid oxidase and its involvement in schizophrenia

D-amino acid oxidase (DAO) is a flavoenzyme that metabolizes certain D-amino acids, notably the endogenous N-methyl D-aspartate receptor (NMDAR) co-agonist, D-serine. As such, it has the potential to modulate the function of NMDAR and to contribute to the widely hypothesized involvement of NMDAR signalling in schizophrenia. Three lines of evidence now provide support for this possibility: DAO shows genetic associations with the disorder in several, although not all, studies; the expression and activity of DAO are increased in schizophrenia; and DAO inactivation in rodents produces behavioural and biochemical effects, suggestive of potential therapeutic benefits. However, several key issues remain unclear. These include the regional, cellular and subcellular localization of DAO, the physiological importance of DAO and its substrates other than D-serine, as well as the causes and consequences of elevated DAO in schizophrenia. Herein, we critically review the neurobiology of DAO, its involvement in schizophrenia, and the therapeutic value of DAO inhibition. This review also highlights issues that have a broader relevance beyond DAO itself: how should we weigh up convergent and cumulatively impressive, but individually inconclusive, pieces of evidence regarding the role that a given gene may have in the aetiology, pathophysiology and pharmacotherapy of schizophrenia?

Potential pathophysiological role of d-amino acid oxidase in schizophrenia: immunohistochemical and in situ hybridization study of the expression in human and rat brain

D-Amino acid oxidase (DAO) is a peroxisomal flavoenzyme that catalyzes oxidative deamination of a wide range of D-amino acids. Among the possible substrates of DAO in vivo, D-serine is proposed to be a neuromodulator of the N-methyl-D-aspartate (NMDA) type glutamate receptor. The gene for DAO was reported to be associated with schizophrenia. Since DAO is expected to be one of the key enzymes in the regulation of NMDA neurotransmission, the modulation of the enzyme activity is expected to be therapeutical for neuronal disorders. In search of the pathophysiological role of DAO, we analyzed the distribution of DAO mRNA and protein in the rat and human brain. In rat, the distribution of DAO mRNA was newly detected in choroid plexus (CP) epithelial cells in addition to glial cells of pons, medulla oblongata, and especially Bergmann glia of cerebellum. Moreover, to investigate how DAO expression level is altered in schizophrenia, we performed immunohistochemistry in the human brain. In agreement with the results in the rat brain, the immunoreactivity for DAO was detected in glial cells of rhombencephalon and in CP. Furthermore, higher level of DAO expression was observed in schizophrenic CP epithelial cells than that in non-schizophrenic cases. These results suggest that an increase in DAO expression in parts of the brain is involved in aberrant D-amino acid metabolism. In particular, gene expression of DAO in CP suggests that DAO may regulate D-amino acid concentration by modulating the cerebrospinal fluid and may be regarded as a potential therapeutic target for schizophrenia.

High Soluble Expression of d-Amino Acid Oxidase in Escherichia coli Regulated by a Native Promoter

To express high-active soluble D-amino acid oxidase (DAAO), a constitutive plasmid that is regulated by a native hydantoinase promoter (P(Hase)), was constructed. A D-amino acid oxidase gene (dao) was ligated with the P(Hase) and cloned into pGEMKT to constitutively express protein of DAAO without the use of any inducer such as isopropyl beta-D-1-thiogalactopyranoside which is poisonous to the cells and environment. The ribosome binding site region, host strain, and fermentation conditions were optimized to increase the expression level. When cultivated in a 5-m(3) fermenter, the enzyme activity of JM105/pGEMKT-R-DAAO grown at 37 degrees C was found to be 32 U/mL and increase 16-fold over cells of BL21(DE3)/pET-DAAO grown at 28 degrees C. These results indicate the success of our approaches to overproducing DAAO in soluble form in Escherichia coli.

Long-Term Treatment With Morphine Increases the D-Serine Content in the Rat Brain by Regulating the mRNA and Protein Expressions of Serine Racemase and D-Amino Acid Oxidase

Recent studies indicate that an endogenous co-agonist for an N-methyl-D-aspartate (NMDA) receptor–related glycine site, D-serine, is synthesized by serine racemase and is metabolized by D-amino acid oxidase (DAO) and that acute treatment with morphine augments the gene expression of serine racemase and DAO in rat brain. To further elucidate the mechanism underlying the activation of NMDA receptors following chronic opioid administration, we have evaluated the effects of the chronic administration of morphine on the mRNA and protein expressions of serine racemase and DAO and on the contents of D-serine in several areas of the rat brain. Repeated administration of morphine for 30 days produced a significant augmentation of both the mRNA and protein expressions of serine racemase in all the brain regions, whereas no significant change in the protein expression of DAO was observed in all the brain regions. Furthermore, the chronic administration caused a slight but significant elevation in the concentration of D-serine in the cortex, striatum, and hippocampus. These results indicate the elevated D-serine level following the chronic morphine treatment could at least in part be involved in the activation of NMDA receptors via the glycine site.

Effects of MK-801 on the expression of serine racemase and d-amino acid oxidase mRNAs and on the d-serine levels in rat brain

We have investigated the acute effects of the increasing doses of non-competitive N-methyl- d-aspartate receptor antagonist MK-801 (0.2–1.6 mg/kg) on the expression of serine racemase and d-amino acid oxidase (DAO) mRNAs in several brain areas of rats. We have also evaluated the effects of the chronic administration of MK-801 (0.4 mg/kg) on the gene expression of serine racemase and DAO and on the d-serine concentrations. A dose-dependent augmentation of the expression of serine racemase mRNA was seen in most brain areas at both 1 and 4 h after the administration. In contrast, a drastic decline in the expression of DAO mRNA was observed in most brain areas 1 h after the MK-801 administration, whereas a dose-dependent elevation in the expression of DAO mRNA was observed in most brain areas 4 h after the administration. The chronic MK-801 administration produced a significant increase in the expression of serine racemase mRNA in almost all brain areas, whereas no significant changes were found in the level of DAO mRNA in most brain areas. In addition, the chronic administration caused a slight but significant elevation in the concentrations of d-serine in the cortex and striatum. These present findings indicate that increasing the serine racemase mRNA and no changes in the DAO mRNA after the chronic administration could contribute to the elevation of the d-serine level in the forebrain, and that serine racemase and DAO could play an important role in the regulation of N-methyl- d-aspartate receptors via the d-serine metabolism.

Changes in D‐serine levels and localization during postnatal development of the rat vestibular nuclei

The patterns of development of the vestibular nuclei (VN) and their main connections involving glutamate neurotransmission offer a good model for studying the function of the glial-derived neuromodulator D-serine in synaptic plasticity. In this study we show that D-serine is present in the VN and we analyzed its distribution and the levels of expression of serine racemase and D-amino acid oxidase (D-AAO) at different stages of postnatal (P) development. From birth to P21, high levels of D-serine were detected in glial cells and processes in all parts of the VN. This period corresponded to high expression of serine racemase and low expression of D-AAO. On the other hand, in the mature VN D-serine displayed very low levels and was mainly localized in neuronal cell bodies and dendrites. This drop of D-serine in adult stages corresponded to an increasing expression of D-AAO at mature stages. High levels of glial D-serine during the first 3 weeks of postnatal development correspond to an intense period of plasticity and synaptogenesis and maturation of VN afferents, suggesting that D-serine could be involved in these phenomena. These results demonstrate for the first time that changes in D-serine levels and distribution occur during postnatal development in the central nervous system. The strong decrease of D-serine levels and the glial-to-neuronal switch suggests that D-serine may have distinct functional roles depending on the developmental stage of the vestibular network.

Potential Role for Astroglial d-Amino Acid Oxidase in Extracellular d-Serine Metabolism and Cytotoxicity

D-amino acid oxidase (DAO) is a flavoenzyme that catalyzes the oxidation of D-amino acids. In the brain, gene expression of DAO is detected in astrocytes. Among the possible substrates of DAO in vivo, D-serine is proposed to be a neuromodulator of the N-methyl-D-aspartate (NMDA) receptor. In a search for the physiological role of DAO in the brain, we investigated the metabolism of extracellular D-serine in glial cells. Here we show that after D-serine treatment, rat primary type-1 astrocytes exhibited increased cell death. In order to enhance the enzyme activity of DAO in cells, we established stable rat C6 glial cells overexpressing mouse DAO designated as C6/DAO. Treatment with a high dose of D-serine led to the production of hydrogen peroxide (H(2)O(2)) followed by apoptosis in C6/DAO cells. Among the amino acids tested, D-serine specifically exhibited a significant cell death-inducing effect. DAO inhibitors, i.e., sodium benzoate and chlorpromazine, partially prevented the death of C6/DAO cells treated with D-serine, indicating the involvement of DAO activity in d-serine metabolism. Overall, we consider that extracellular D-serine can gain access to intracellular DAO, being metabolized to produce H(2)O(2). These results support the proposal that astroglial DAO plays an important role in metabolizing a neuromodulator, D-serine.

P.6.d.021 Histo-and immunochemical detection of inducible NOS and TNF-alpha in the locus coeruleus of human opiate addicts

Spinal d-amino acid oxidase (DAAO) is an FAD-dependent peroxisomal flavoenzyme which mediates the conversion of neutral and polar d-amino acids (including d-serine) to the corresponding α-keto acids, and simultaneously produces hydrogen peroxide and ammonia. This study has aimed to explore the potential contributions of spinal DAAO and its mediated hydrogen peroxide/d-serine metabolism to the development of morphine-induced hyperalgesia. Bi-daily subcutaneous injections of morphine to mice over 7 days induced thermal hyperalgesia as measured by both the hot-plate and tail-immersion tests, and spinal astroglial activation with increased spinal gene expression of DAAO, glial fibrillary acidic protein (GFAP) and pro-inflammatory cytokines (interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)). Subcutaneous injections of the potent DAAO inhibitor CBIO (5-chloro-benzo[d]isoxazol-3-ol) prevented and reversed the chronic morphine-induced hyperalgesia. CBIO also inhibited both astrocyte activation and the expression of pro-inflammatory cytokines. Intrathecal injection of the hydrogen peroxide scavenger PBN (phenyl-N-tert-butylnitrone) and of catalase completely reversed established morphine hyperalgesia, whereas subcutaneous injections of exogenous d-serine failed to alter chronic morphine-induced hyperalgesia. These results provided evidence that spinal DAAO and its subsequent production of hydrogen peroxide rather than the d-serine metabolism contributed to the development of morphine-induced hyperalgesia.

MK-801 upregulates the expression of d-amino acid oxidase mRNA in rat brain

We have evaluated the effect of the systemic administration of MK-801 (0.4 mg/kg) on the gene expression of d-amino acid oxidase (DAO) in several brain areas of the rat. The levels of DAO mRNA in all the brain areas significantly increased and peaked at 4 h after the administration. The present results suggest that there is a link between the expression of DAO mRNA and the N-methyl- d-aspartate (NMDA) receptor activity.

Regulation of D-amino acid oxidase expression in the yeast Rhodotorula gracilis.

Rhodotorula gracilis is a oleaginous yeast which utilizes D-amino acids as a source of carbon and/or nitrogen. D-amino acid oxidase (DAAO), which converts D-amino acids in the corresponding alpha-keto acids and ammonia, is the first enzyme involved in the catabolism of D-amino acids. DAAO activity is induced by the presence of D-alanine, but the presence of the L-isomer prevents induction by inhibiting the transport of D-alanine into cells. To understand how DAAO expression is regulated, R. gracilis cells were grown on media containing different nitrogen and/or carbon sources. As a general rule, the level of DAAO mRNA reached a maximum after 15 h growth and preceded by approximately 6 h the maximum level of DAAO activity. The inducer D-alanine acts by increasing the rate of DAAO mRNA transcription: the increase in DAAO expression is due essentially to de novo synthesis. The presence of a supplemental carbon source (e.g. succinate or glucose) does not repress DAAO expression. Ammonium sulphate appears to have a negative effect on DAAO mRNA translation and on the expression of DAAO activity: DAAO is only partially active when the yeast is grown in the presence of D-alanine and ammonium sulphate. The best expression of DAAO activity was obtained by growing the cells for 12 h at 30 degrees C in the presence of glucose and D-alanine using cells pre-cultured for 10 h on glucose and L-alanine (0.99 U/mg protein, corresponding to approximately 1.0% total proteins in the crude extract). Under these growth conditions a six-fold increase in DAAO production was achieved.

High expression of Trigonopsis variabilisd-amino acid oxidase in Pichia pastoris

To explore a new approach of high expression of d-amino acid oxidase (DAAO) in Pichia pastoris, a gene encoding DAAO from Trigonopsis variabilis ( TvDAAO gene) deleted intron was prepared by PCR amplification and cloned into the intracellular expression vector pPIC3.5K. The expression plasmid pPIC3.5K-DAAO linearized by SalI was transformed into Pichia pastoris strain GS115 ( his − mut +). By means of MM and MD plates and PCR, the recombinant P. pastoris strains ( his + mut +) were obtained. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant strain PD27 with the highest expression of DAAO was screened through activity assay and its high-density fermentation was carried out in a 1-l fermentor. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant cells with high expression of DAAO were screened and the high-density fermentation was carried out in a 1-l fermentor. Interestingly, the DAAO expression level reached up to 473 U/g dry cell weight in fermentation yield. Finally, 1-hexanol was used to break recombinant cells and the specific activity of DAAO was 1.46 U/mg protein in crude extraction.

Gene expression of D-amino acid oxidase in cultured rat astrocytes: regional and cell type specific expression

Neuromodulative free D-serine is present in mammalian brain, and localized to type-2 astrocytes in culture. D-amino acid oxidase (DAO) is a flavoenzyme that catalyzes D-amino acids. We examined the DAO gene expression in cultured rat astrocytes by reverse transcriptase-polymerase chain reaction. We established a method to prepare highly purified culture of type-1 and type-2 astrocytes from any brain region. This method utilizes combination of cell type specific separation by shaking and subsequent purification by immunopanning or treatment with cytosine arabinoside. We detected higher DAO gene expression in type-1 astrocyte cultures from cerebellum than that from cerebral cortex. In cerebellum, we observed higher DAO expression in type-1 astrocyte cultures than that in type-2. We also revealed that DAO expression in C6, corresponding to type-1 astrocyte, was higher than that in CG-4 derived type-2 astrocytes.

Development of tailor-made glycidyl methacrylate-divinyl benzene copolymer for immobilization of d-amino acid oxidase from Aspergillus species strain 020 and its application in the bioconversion of cephalosporin C

A tailor-made glycidyl methacrylate-divinyl benzene (GMA-DVB) copolymer PC-3 was evolved by studying the effect of synthesis variables on binding and expression of d-amino acid oxidase (DAAO) from Aspergillus species strain 020. Almost quantitative binding (100%) and a high yield of immobilization per unit of enzyme loaded was achieved. Optimum pH, optimum temperature and K m for soluble and IME-DAAO preparations were 8.0–8.5 and 8.0; 30 and 37°C; and 0.73 and 2.78 mM for ceph C, respectively. The developed immobilized DAAO was used for the production of glutaryl-7-aminocephalosporanic acid from cephalosporin C (ceph C). A high conversion of >95% was achieved by using 3% (w/v) solution of ceph C, 48 U of DAAO per g of ceph C, keeping dissolved oxygen level above 50%, maintaining the pH between 7.6 and 7.8 and temperature at 24°C. The immobilized DAAO was used for 60 cycles in a stirred tank reactor.

内耳における神経情報伝達

Fast excitatory synaptic transmission in the inner ear has been proposed to be mediated by an excitatory amino acid, possibly L-glutamate, and its receptors. Previous studies have revealed that certain types of ionotropic glutamate receptors are expressed in various combinations of subtypes in the cochlea and vestibule. Other studies have suggested that some glutamate receptors may have non-neural functions, such as cell differentiation and migration. Recently the glutamate receptor a subfamily has been identified, although little is known about its physiological characteristics thus far. Since 1997 it has been known that the 61 subunit is highly expressed in hair cells of auditory and vestibular systems, which suggests that the 61 subunit may play an important functional role in these systems. The expression of NMDA receptor mRNA in spiral ganglion cells has also been demonstrated, suggesting involvement of the NMDA receptor in neurotransmission in the inner ear. In search of coagonists of the NMDA receptor other than glycine, D-serine has been found to be present in mammalian central nervous systems and is regarded as a possible neuro-modulator of the NMDA receptor. We have demonstrated the gene expression of D-amino acid oxidase (DAO) in the rat temporal bone using RT-PCR analysis. This observation suggests that D-serine, which is the substrate of DAO, may exist in the inner ear and may modulate the neurotransmission of NMDA receptor subunit NR1.

Cloning, sequencing and expression in E. coli of a d-amino acid oxidase cDNA from Rhodotorula gracilis active on cephalosporin C

We have cloned the cDNA coding for the Rhodotorula gracilis d-amino acid oxidase (DAAO), an enzyme that performs with high catalytic efficiency biotechnologically relevant bioconversions, by PCR amplification. The first strand cDNA was synthesised from the total mRNA fraction isolated from R. gracilis cells grown under DAAO-inducing conditions. The R. gracilis DAAO cDNA consists of 1104 bp encoding a protein of 368 amino acids. The insertion of the cDNA into the pKK223-3 plasmid allowed the expression of recombinant DAAO in Escherichia coli as a wholly soluble and catalytically active holoenzyme (≈0.5 U mg −1 protein) with a fermentation yield, in terms of DAAO units, of 800 U 1 −1. This level of expression allowed the purification, in homogeneous form and high yield (50%), of the recombinant enzyme which showed a high catalytic activity on cephalosporin C as substrate. The nucleotide sequence reported in this paper will appear in the GenBank nucleotide sequence databases under accession number U60066.

Gene Expression of D-Amino Acid Oxidase in Rabbit Kidney1

Although D-amino acid oxidase (DAO) [EC 1.4.3.3] activity in rabbit kidney extract was undetectable, protein immunoreactive toward rabbit anti-pig kidney DAO antiserum and RNAs that hybridized with fragments of human and pig DAO cDNAs were detected distinctly in the rabbit kidney. A cDNA clone, RD22, was isolated from the rabbit kidney cDNA library by hybridization with a fragment of human DAO cDNA. Analysis of the nucleotide sequence revealed a 2,018 nucleotide sequence encoding a protein consisted of 347 amino acids. The number of amino acid residues was identical with those of human and pig DAOs, and the amino acid sequence showed 80 and 83% identity with pig and human DAOs, respectively. RNAs that hybridized with RD22 DNA fragment also existed in rabbit kidney, and their sizes were the same as those of the RNAs detected with the human and pig DAO cDNA fragments. RD22-derived protein was hardly synthesized by an in vitro expression system. However, a cDNA fragment lacking most of the 5'-untranslated region and its mutants containing base changes around the initiation codon did direct protein synthesis. Moreover, the protein derived from the partial cDNA fragment containing a large part of the coding region sequence showed immunoreactivity toward anti-pig DAO antiserum. The results suggest that one of the causes of the very poor synthesis of DAO protein in rabbit kidney is translational suppression in the synthetic process.

In vivo and in vitro expression of porcine D-amino acid oxidase: in vitro system for the synthesis of a functional enzyme.

In vivo expression of D-amino acid oxidase (EC 1.4.3.3, DAO), one of the principal and characteristic enzymes of the peroxisomes of porcine kidney, was examined by use of cloned complementary DNA [Fukui, K., Watanabe, F., Shibata, T., & Miyake, Y. (1987) Biochemistry 26, 3612-3618]. RNA blot hybridization analysis revealed that DAO is expressed abundantly in kidney and liver, is expressed significantly in brain, but is not expressed in lung of pig. Three mRNA species were expressed in kidney and liver, but only one was detected in brain. These results show the presence of tissue-specific regulation of DAO gene expression. In vitro expression of a functional enzyme was achieved through the construction of a recombinant plasmid containing an SP6 promoter and a restriction enzyme fragment of cDNA to generate a DAO-specific RNA transcript. The in vitro translation product of the capped RNA transcript showed significant catalytic activity, which was inhibited strongly by benzoate, a potent inhibitor of DAO. The kinetic properties of the in vitro synthesized enzyme were comparable to those of the purified enzyme from porcine kidney. It is now possible to synthesize a functional D-amino acid oxidase in vitro and to investigate its structure-function relationships.