PEG-DAAO conjugate: A promising tool for cancer therapy optimized by protein engineering.

The flavoenzyme D-amino acid oxidase (DAAO) represents a potentially good option for cancer enzyme prodrug therapy as it produces H2O2 using D-amino acids as substrates, compounds present at low concentration in vivo and that can be safely administered to regulate H2O2 production. We optimized the cytotoxicity of the treatment by: i) using an efficient enzyme variant active at low O2 and D-alanine concentrations (mDAAO); ii) improving the stability and half-life of mDAAO and the enhanced permeability and retention effect by PEGylation; and iii) inhibiting the antioxidant cellular system by a heme oxygenase-1 inhibitor (ZnPP). A very low amount of PEG-mDAAO (10 mU, 50 ng of enzyme) induces cytotoxicity on various tumor cell lines. Notably, PEG-mDAAO seems well suited for in vivo evaluation as it shows the same cytotoxicity at air saturation (21%) and 2.5% O2, a condition resembling the microenvironment found in the central part of tumors.

Selenoprotein W as a molecular target of d-amino acid oxidase is regulated by d-amino acid in chicken neurons.

Selenoprotein W (SelW) is an important member of the avian selenoprotein family. It is well known for its important role in protecting neurons from oxidative stress during neuronal development. d-Amino acid (d-serine), as a neurotransmitter in the central nervous system (CNS), can mediate neurotoxicity. d-Amino acid oxidase (DAAO) is responsible for regulating the d-serine levels in cells. However, the correlation between SelW and DAAO is not clear yet. To investigate the regulations between SelW and DAAO, chicken embryo monolayer neurons were treated with d-serine and/or Se. In this study, we predicted molecular binding between SelW and DAAO. These results showed that the 9-16, 18, 41-47 and 66 residues of SelW could combine with the DAAO, which suggested that chicken SelW might be the target of DAAO. We determined the DAAO activity and the mRNA expression of SelW in in vitro cultured chicken embryo primitive neuron cells. d-Serine influenced the activity of DAAO and, moreover, a significant increase in the mRNA expression of SelW was found in neurons treated with Se. Notably, we also observed changes in the expression of SelW and DAAO when neurons were treated with various concentrations of d-serine and Se. In conclusion, these data suggest that d-serine could regulate the mRNA expression of SelW by interfering with the activity of DAAO in chicken embryo neurons.

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.

Optimization of D-amino acid oxidase for low substrate concentrations--towards a cancer enzyme therapy.

D-amino acid oxidase (DAAO) has recently become of interest as a biocatalyst for industrial applications and for therapeutic treatments. It has been used in gene-directed enzyme prodrug therapies, in which its production of H2O2 in tumor cells can be regulated by administration of substrate. This approach is limited by the locally low O2 concentration and the high K(m) for this substrate. Using the directed evolution approach, one DAAO mutant was identified that has increased activity at low O2 and D-Ala concentrations and a 10-fold lower K(m) for O2. We report on the mechanism of this DAAO variant and on its cytotoxicity towards various mammalian cancer cell lines. The higher activity observed at low O2 and D-Ala concentrations results from a combination of modifications of specific kinetic steps, each being of small magnitude. These results highlight the potential in vivo applicability of this evolved mutant DAAO for tumor therapy.

Molecular cloning and expression in Escherichia coli of an active fused Zea mays L. D-amino acid oxidase.

D-Amino acid oxidase (DAAO) is an FAD-dependent enzyme that metabolizes D-amino acids in microbes and animals. However, such ability has not been identified in plants so far. We predicted a complete DAAO coding sequence consisting of 1158 bp and encoding a protein of 386 amino acids. We cloned this sequence from the leaf cDNA population of maize plants that could utilize D-alanine as a nitrogen source and grow normally on media containing D-Ala at the concentrations of 100 and 1000 ppm. For more understanding of DAAO ability in maize plant, we produced a recombinant plasmid by the insertion of isolated cDNA into the pMALc2X Escherichia coli expression vector, downstream of the maltose-binding protein coding sequence. The pMALc2X-DAAO vector was used to transform the TB1 strain of E. coli cells. Under normal growth conditions, fused DAAO (with molecular weight of about 78 kDa) was expressed up to 5 mg/liter of bacterial cells. The expressed product was purified by affinity chromatography and subjected to in vitro DAAO activity assay in the presence of five different D-amino acids. Fused DAAO could oxidize D-alanine and D-aspartate, but not D-leucine, D-isoleucine, and D-serine. The cDNA sequence reported in this paper has been submitted to EMBL databases under accession number AM407717.

Inhibition of D-amino-Acid oxidase activity induces pain relief in mice.

(1). We investigated the effects of inhibiting D: -amino-acid oxidase (DAO) activity on nociceptive responses through the use of mutant ddY/DAO(-) mice, which lack DAO activity, and through the application of a selective inhibitor of DAO, sodium benzoate, in the tail flick test, hot-plate test, formalin test, and acetic acid-induced writhing test. (2). Compared with normal ddY/DAO+ mice, ddY/DAO(- )mice showed significantly prolonged tail withdrawal latency in the tail flick test and licking/jumping latency in the hot-plate test, as well as significantly reduced duration of licking/biting in the late phase of the formalin test and the number of abdominal writhing in the acetic acid-induced writhing test. (3). In addition, we investigated the effects of sodium benzoate in Kunming mice having normal DAO activity. (4). Intravenous administration of sodium benzoate (400 mg/kg) significantly inhibited pain responses of the late phase of the formalin test and abdominal writhing responses in the acetic acid-induced writhing test, with no effects on the early phase flinch responses in the formalin test, nociceptive responses in the tail flick test, or hot-plate test. (5). These results suggest that DAO acts as a pro-nociceptive factor in pain, particularly chronic pain, transmission and modulation, and may be a target for pain treatment.

Alanine catabolism in Klebsiella aerogenes: molecular characterization of the dadAB operon and its regulation by the nitrogen assimilation control protein.

Klebsiella aerogenes strains with reduced levels of D-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of nitrogen. The alanine catabolic operon of Klebsiella aerogenes, dadAB, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of dadA strains. The operon has a high similarity to the dadAB operon of Salmonella typhimurium and the dadAX operon of Escherichia coli, each of which codes for the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for E. coli and S. typhimurium, the dad operon of K. aerogenes is activated by the Ntr system, mediated in this case by the nitrogen assimilation control protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position -44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein.

Involvement of D-amino-acid oxidase in D-amino acid utilization in the mouse.

Mutant ddY/DAO- mice lacking D-amino-acid oxidase were examined for their ability to utilize D-phenylalanine in place of its L-isomer. Chemically defined milk powder devoid of phenylalanine was used as a basal diet. Adult ddY/DAO- mice and normal ddY/DAO+ mice having D-amino-acid oxidase lost weight every day when fed this diet. However, they maintained their weight when fed the milk powder diet supplemented with 0.33% L-phenylalanine. Although adult ddY/DAO+ mice maintained their weight when fed the milk powder diet supplemented with 0.33% D-phenylalanine, ddY/DAO- mice lost weight while feeding on this diet, which indicated that the ddY/DAO- mice could not utilize D-phenylalanine in place of its L-isomer. Both ddY/DAO- and ddY/DAO+ mice maintained their weight when feeding on the milk powder diet enriched with 0.41% sodium phenylpyruvate, which suggested to us that the ddY/DAO- mice had the ability to invert this alpha-keto acid to L-phenylalanine. Therefore, these results indicate that D-amino-acid oxidase is indispensable for D-amino acid utilization. This is consistent with the concept that D-amino acids are oxidatively deaminated to the corresponding alpha-keto acids by D-amino-acid oxidase and that these keto intermediates are then asymmetrically reaminated to their L-amino acids.