Characterization of the Enzymatic and Structural Properties of Human D-Aspartate Oxidase and Comparison with Those of the Rat and Mouse Enzymes

Abstract

D-Aspartate (D-Asp), a free D-amino acid found in mammals, plays crucial roles in the neuroendocrine, endocrine, and central nervous systems. Recent studies have implicated D-Asp in the pathophysiology of infertility and N-methyl-D-Asp receptor-related diseases. D-Asp oxidase (DDO), a degradative enzyme that is stereospecific for acidic D-amino acids, is the sole catabolic enzyme acting on D-Asp in mammals. Human DDO is considered an attractive therapeutic target, and DDO inhibitors may be potential lead compounds for the development of new drugs against the aforementioned diseases. However, human DDO has not been characterized in detail and, although preclinical studies using experimental rodents are prerequisites for evaluating the in vivo effects of potential inhibitors, the existence of species-specific differences in the properties of human and rodent DDOs is still unclear. Here, the enzymatic activity and characteristics of purified recombinant human DDO were analyzed in detail. The kinetic and inhibitor-binding properties of this enzyme were also compared with those of purified recombinant rat and mouse DDOs. In addition, structural models of human, rat, and mouse DDOs were generated and compared. It was found that the differences among these DDO proteins occur in regions that appear involved in migration of the substrate/product in and out of the active site. In summary, detailed characterization of human DDO was performed and provides useful insights into the use of rats and mice as experimental models for evaluating the in vivo effects of DDO inhibitors.

Among the free D-amino acids found in mammals, D-serine (D-Ser) and D-aspartate (D-Asp) have been studied most extensively. D-Ser persists at high concentrations throughout the life of an animal and is concentrated predominantly in the mammalian forebrain. D-Ser binds to the glycine-binding site of the N-methyl-D-Asp (NMDA) receptor, a subtype of the L-glutamate (L-Glu) receptor family, and potentiates glutamatergic neurotransmission in the central nervous system.^1,2) Astroglial-derived D-Ser regulates NMDA receptor-dependent long-term potentiation and depression in hypothalamic and hippocampal excitatory synapses.^3,4) These lines of evidence suggest that D-Ser plays an important role in the regulation of brain functions by acting as a co-agonist for the NMDA receptor. Indeed, perturbation of D-Ser levels in the nervous system has recently been implicated in the pathophysiology of various neuropsychiatric disorders, including schizophrenia,^5–8) Alzheimer’s disease,^9,10) and amyotrophic lateral sclerosis.^11,12)

Unlike the tissue-specific expression of D-Ser, substantial amounts of free D-Asp are present in a wide variety of mammalian tissues and cells, particularly those of the central nervous, neuroendocrine, and endocrine systems. Several lines of evidence suggest that D-Asp plays an important role in regulating developmental processes, hormone secretion, and steroidogenesis.^13–15) The amounts of D-Asp in human seminal plasma and spermatozoa are significantly lower in oligoasthenoteratospermic and azoospermic donors than normospermic donors.¹⁶⁾ Furthermore, in female patients undergoing in vitro fertilization, the D-Asp content of pre-ovulatory follicular fluid is lower in older patients than in younger patients; this decrease in D-Asp content appears to reflect a reduction in oocyte quality and fertilization competence.¹⁷⁾ Overall, current evidence suggests that decreases in D-Asp levels may be involved in the pathophysiology of infertility. Furthermore, D-Asp stimulates the NMDA receptor by acting as an agonist that binds to the L-Glu-binding site of the receptor.^18,19) Recent studies have suggested that, similar to D-Ser, D-Asp acts as a signaling molecule in nervous and neuroendocrine systems, at least in part, by binding to the NMDA receptor, and plays an important role in the regulation of brain functions.^14,15,20) In support of this proposal, it was reported recently that D-Asp levels in the prefrontal cortex and striatum of post-mortem brains of schizophrenic patients are significantly lower than those of non-psychiatrically ill individuals.²¹⁾

In mammalian tissues, two types of degradative enzymes that are stereospecific for D-amino acids have been identified, namely, D-amino acid oxidase (DAO, also abbreviated as DAAO; EC 1.4.3.3) and D-Asp oxidase (DDO, also abbreviated as DASPO; EC 1.4.3.1). DAO and DDO are FAD-containing flavoproteins that catalyze the oxidative deamination of D-amino acids to generate 2-oxo acids along with hydrogen peroxide and ammonia.^22,23) DAO displays broad substrate specificity and acts on several neutral and basic D-amino acids, including D-Ser. On the other hand, DDO is highly specific for acidic D-amino acids, such as D-Asp and D-Glu, none of which are substrates of DAO. DAO and DDO have both been identified in various organisms and their physiological roles in vivo are being investigated extensively. Mammalian DAO and DDO are presumed to regulate the levels of several endogenous and exogenous D-amino acids, including D-Ser and D-Asp, in various organs^24,25); however, their physiological roles in vivo have yet to be clarified fully.

As described above, reduced levels of D-Asp in the nervous system and a resulting dysfunction in NMDA receptor-mediated neurotransmission is thought to occur during the onset of various mental disorders, including schizophrenia^14,15,20,21); hence, a substance capable of increasing D-Asp levels and activating NMDA receptor function may present a novel foundation for the development of antipsychotic drugs. One way to increase the level of D-Asp is to prevent its metabolic degradation by DDO. Indeed, DDO-deficient mice have elevated concentrations of D-Asp in several brain regions and exhibit specific antidepressant and antischizophrenic behaviors.^26,27) These findings support the concept that DDO inhibitors that activate NMDA receptor function by augmenting the levels of D-Asp in the brain would be new and useful antipsychotic drugs for the treatment of NMDA receptor-related diseases. DDO inhibitors may also be lead compounds for the development of new drugs to treat infertility since D-Asp is thought to be involved in the quality control of germ cells.^16,17) Overall, human DDO is an attractive therapeutic target and several compounds that inhibit the activity of mammalian DDO in vitro have been identified to date; however, the inhibitory potency of these compounds is limited.^28–33)

Preclinical studies using experimental rodents are essential prerequisites for evaluating the in vivo effects of clinically useful enzyme inhibitors. However, species-related differences in some properties of target enzymes occur frequently and can lead to inaccurate evaluation of the in vivo effects of the inhibitors; therefore, it is vital to characterize and compare the properties of human enzymes with those of their rodent homologs. The cDNA encoding human DDO was initially cloned from human brain and subsequently from human hepatocellular carcinoma Hep G2 cells.^34,35) These cDNAs were successfully expressed in Escherichia coli, and some properties of the purified recombinant human DDO proteins have been investigated. Specifically, the absorption spectra, the apparent kinetic parameters for acidic D-amino acids, and the FAD binding affinity of human DDO have been reported^31,34,35); however, details of the enzymatic properties of this protein have not yet been elucidated. The structural features of the human DDO protein are also still unclear, mainly because the three-dimensional (3D) structure of mammalian DDO has not yet been reported.

Here, purified recombinant human DDO was enzymatically characterized in detail. The kinetic and inhibitor binding properties of this enzyme were also compared with those of purified recombinant rat and mouse DDOs. In addition, predicted structural models of human, rat and mouse DDOs were generated and compared. Overall, this study describes a detailed characterization of human DDO and provides useful insights into the use of rats and mice as experimental models for evaluating the in vivo effects of DDO inhibitors.

MATERIALS AND METHODS

Chemicals

Ampicillin, proteinogenic L-amino acids and their enantiomers, N-methyl-L-Asp, NMDA, N-methyl-L-Glu, bovine serum albumin, o-dianisidine, and catalase from Aspergillus niger were purchased from Sigma-Aldrich (St Louis, MO, U.S.A.). N-Methyl-D-Glu was purchased from Bachem (Bubendorf, Switzerland). Aminooxyacetic acid, Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin were purchased from Nacalai Tesque (Kyoto, Japan). Isopropyl-β-D-thiogalactopyranoside, imidazole, FAD, 2,4-dinitrophenylhydrazine, horseradish peroxidase, malonate, and meso-tartrate were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Fetal bovine serum was purchased from Gibco-BRL (Gaithersburg, MD, U.S.A.). The purity of N-methyl-L-Asp was greater than 99% as verified using thin layer chromatography by the vendor. All other chemicals used were of the highest grade available and were purchased from commercial sources.

Isolation of the Rat Ddo cDNA Clone from NRK-52E Cells

Rat kidney NRK-52E cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37°C in 5% CO₂/95% air. Total RNA was extracted from the cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. For first-strand cDNA synthesis, the RNA samples (5 µg) were reverse-transcribed in a 20 µL reaction mixture containing 200 units of SuperScript III Reverse Transcriptase and 0.5 µg of oligo(dT)_12–18 primer (Invitrogen, Carlsbad, CA, U.S.A.). After incubation at 50°C for 1 h, 1 µL (2 units) of RNaseH (Invitrogen) was added and the mixture was incubated at 37°C for 20 min. Based on the sequence of the rat Ddo gene deposited in the National Center for Biotechnology Information database (NCBI Gene ID: 685325), the following primers were designed: 5′-AGA TCT ATG GAC ACG GTG CGT ATT GCG GTC-3′ (forward primer) and 5′-GAA TTC CTA CAG CTT TGA TAG GGA AGC TGG GG-3′ (reverse primer). Restriction enzyme (BglII and EcoRI) sites were included at the 5′-ends of the forward and reverse primers, respectively. The rat Ddo cDNA was amplified by polymerase chain reaction using these primers and the first-strand cDNA as a template. The reaction product was cloned into the pT7Blue vector (Novagen, Madison, WI, U.S.A.) to generate pT7-rDDO, and its sequence was confirmed.

Construction of Expression Plasmids

The construction of expression plasmids containing N-terminally His-tagged human and mouse DDO (pRSET-His-hDDO and pRSET-His-mDASPO, respectively) has been described previously.^30,34) The 1 kb BglII–EcoRI fragment of pT7-rDDO containing the entire rat Ddo coding sequence was subcloned into the pRSET-B vector (Invitrogen) to generate the N-terminally His-tagged rat DDO expression plasmid (pRSET-His-rDDO).

Expression and Purification of Recombinant Proteins

E. coli BL21(DE3)pLysS cells were transformed with expression plasmids and cultured at 37°C with shaking in Luria–Bertani medium containing 100 µg/mL ampicillin. Crude extracts were prepared from cells transformed with pRSET-His-hDDO and pRSET-His-mDASPO, as described previously.^33,36) For cells transformed with pRSET-His-rDDO, the culture was grown to A₆₂₀=0.5 and then incubated for an additional 30 min at a reduced temperature (26°C). After the addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside, the culture was incubated at 26°C for a further 16 h. The cells were then centrifuged at 10000×g for 10 min at 4°C, and crude extract was prepared using BugBuster Protein Extraction Reagent and Lysonase Bioprocessing Reagent (Novagen) in the presence of 50 µM FAD and protease inhibitors (Nacalai Tesque), according to the manufacturer’s instructions.

All recombinant proteins were purified by affinity chromatography using a chelating column. Crude extracts were applied to a His GraviTrap column (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, U.S.A.) equilibrated with 20 mM sodium phosphate buffer (pH 7.4) containing 0.5 M NaCl and 10 mM imidazole. The column was then washed with the same buffer and bound proteins were eluted using a step-wise gradient of 50–500 mM imidazole. Each fraction (2 mL) containing recombinant protein was dialyzed at 4°C for 1 d against 1 L of 10 mM sodium pyrophosphate buffer (pH 8.3) containing 5 mM 2-mercaptoethanol and 10% (v/v) glycerol. The buffer was changed once during dialysis. The dialyzed fractions were recovered and centrifuged at 10000×g for 10 min at 4°C to pellet the proteins denatured during dialysis. The supernatants were recovered as purified enzyme and used immediately for enzyme assays or stored at −80°C until use. All recombinant proteins were purified to near homogeneity when examined by SDS-polyacrylamide gel electrophoresis. The protein concentrations of the purified enzyme preparations were determined using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, U.S.A.), with bovine serum albumin as a standard.

Enzymatic Activity Assays

The activity of DDO was determined using a colorimetric assay for 2-oxo acid production, as described previously.³⁷⁾ Briefly, the standard assay mixture contained appropriate amounts (0.095–1 µg) of the purified enzyme, air-saturated 40 mM sodium pyrophosphate buffer (pH 8.3), 50 ng/µL A. niger catalase, 60 µM FAD, and 20 mM D-Asp in a final volume of 150 µL. Unless otherwise noted, the reaction mixture was incubated at 37°C for 10 min, and then 10 µL of 100% (w/v) trichloroacetic acid was added to stop the reaction. The 2-oxo acid product was reacted with 2,4-dinitrophenylhydrazine and quantified by measuring the A₄₄₅ against a blank mixture lacking D-Asp.

To examine the effect of pH on the activity of DDO towards D-Asp, the enzymatic reaction was performed in the following buffers: 150 mM GTA buffer (50 mM 3,3-dimethylglutaric acid, 50 mM Tris, and 50 mM 2-amino-2-methyl-1,3-propanediol) (pH 5.0–7.5); 40 mM sodium pyrophosphate buffer (pH 7.0–10.0); 40 mM glycine–NaOH buffer (pH 10.0–11.0); 40 mM Na₂HPO₄–NaOH buffer (pH 11.0–12.0); or KCl–NaOH buffer (pH 12.0–13.0). To examine the effect of temperature on the enzymatic activity, the reaction was performed at 25–90°C. To examine the effect of temperature on the stability of DDO, the enzyme was preincubated for 30 min at 30, 40, 45, 50, 55, 60 or 70°C prior to the addition of D-Asp. After the preincubation, D-Asp was added to the mixture and the enzymatic activity was measured as described above. To examine the effect of pH on the stability of DDO, the enzyme was preincubated at 45°C for 30 min with the following buffers prior to the addition of D-Asp: 40 mM sodium pyrophosphate buffer (pH 7.5–9.5) and 40 mM Na₂HPO₄–NaOH buffer (pH 11.0–12.0). After the preincubation, D-Asp was added to the mixture and the enzymatic activity was measured as described above. To examine the effects of various compounds on the activity of DDO, each compound was added to the reaction mixture individually and its relative inhibitory activity was normalized to that of the enzyme alone. The IC₅₀ values of the compounds tested were determined using the following formula, as described previously³³⁾: IC₅₀=10^{(log[A/B]×[50−C]/[D−C]+log B)}, where A and B are the highest and lowest concentrations closest to the middle of the curve, and C and D are the inhibition percentages at B and A, respectively. To determine the maximal velocity (V_max) and Michaelis constant (K_m) values for D-Asp, different final concentrations (1–20 mM) of D-Asp were used as the substrate and the enzymatic reaction was performed under conditions in which the production of 2-oxo acids was linear with the incubation time and exhibited Michaelis–Menten-type properties. The data obtained were fitted to the Michaelis–Menten equation, and the V_max and K_m values were estimated using the nonlinear least-squares fitting algorithm in pro Fit 6.1 software (Quantum soft, Zürich, Switzerland; http://www.quansoft.com). The molecular activity (k_cat) values were calculated from the V_max values and the estimated molecular masses of the recombinant proteins (41,692, 41,700, and 42,074 Da for N-terminally His-tagged human, rat, and mouse DDO, respectively). The inhibitor constant (K_i) values of several compounds were determined by the methods of Cheng and Prusoff³⁸⁾ and Cer et al.³⁹⁾

The enzymatic activity of DDO was also determined using a colorimetric assay for hydrogen peroxide production, based on the method described by Lockridge et al.⁴⁰⁾ In this method, the production of hydrogen peroxide by DDO is coupled to the oxidation of o-dianisidine catalyzed by horseradish peroxidase. Appropriate amounts (0.036–1 µg) of the purified DDO protein were added to a reaction mixture containing air-saturated 40 mM sodium pyrophosphate buffer (pH 8.3), 20 ng/µL horseradish peroxidase, 0.32 mM Triton-stabilized o-dianisidine (a fresh stock solution was prepared daily by mixing 0.8 mL of 10 mM o-dianisidine with 0.2 mL of 20% [v/v] Triton X-100), and 20 mM amino acid in a final volume of 250 µL. When the enzymatic activities of DDO towards D- and L-Tyr were examined, these amino acids were used at 0.8 mM because of their limited solubility. The reaction mixture was incubated at 37°C for 10 min, and then boiled for 10 min to stop the reaction. The solution was cleared by centrifugation at 20000×g for 10 min at 4°C, followed by incubation at room temperature for 10 min. The A₄₆₀ of the supernatant was measured against a blank mixture lacking amino acid or enzyme. The specific activity of the enzyme was calculated on the basis of a molar extinction coefficient at 460 nm of the o-dianisidine oxidation product equal to 11.6 mM⁻¹ cm⁻¹.⁴¹⁾

Computational Modeling of the 3D Structures of DDO and the Human DDO/Malonate Complex

Structural models of the human, rat and mouse DDO proteins were generated using the homology modeling method and SWISS-MODEL on the ExPASy web server (http://swissmodel.expasy.org/).⁴²⁾ The 3D X-ray crystallographic structure of pig DAO (Protein Data Bank ID: 1KIF) was used as the template structure.⁴³⁾ Energy minimization of the structural models was performed using the Steepest Descent minimization algorithm in Swiss-PdbViewer 4.0 software (Swiss Institute of Bioinformatics, Lausanne, Switzerland; http://www.expasy.org/spdbv/).⁴⁴⁾ To determine a theoretical model of the human DDO/malonate complex, the 3D structure of malonate was built using MarvinSketch 5.4 software (ChemAxon, Budapest, Hungary; http://www.chemaxon.com/) by generating low energy tautomeric, stereoisomeric, and ionization states at pH 8.3. Docking of the resulting 3D structure of malonate to the target human DDO protein (prepared as described above) was achieved using AutoDock 4.2 software (The Scripps Research Institute, La Jolla, CA, U.S.A.; http://autodock.scripps.edu/),⁴⁵⁾ and the best docked pose with the lowest energy conformation was selected.

RESULTS AND DISCUSSION

Effects of pH and Temperature on the Activity and Stability of Human DDO

First, the effect of pH on the enzymatic activity of human DDO towards D-Asp was examined. The activity increased gradually as the pH was increased from 6.0 to 8.3, remained approximately constant at pH 8.3 to 12.5, and decreased rapidly when the pH was greater than 12.5 (Fig. 1A). No enzymatic activity was detected at very low and high pH levels (5.0 and 13.0, respectively). Thus, human DDO exhibited a relatively higher activity at alkaline pH levels ranging from pH 8.3 to 12.5. This pH profile of human DDO is markedly different to the previously reported pH-dependence of pig DDO, whose activity is approximately constant at pH 7.5 to 9.0 but decreases at higher pH values.⁴⁶⁾ Subsequently, the effect of temperature on the enzymatic activity of human DDO towards D-Asp was examined at pH 8.3. Under these conditions, human DDO showed maximum activity at 45°C (Fig. 1B).

Fig. 1. The Effects of pH and Temperature on the Activity and Stability of Human DDO

(A) The enzymatic activity of human DDO towards D-Asp at various pH levels. The buffers used for each pH range are described in Materials and Methods. (B) The enzymatic activity of human DDO towards D-Asp at various temperatures and pH 8.3. (C) The effect of a 30 min preincubation of human DDO at pH 8.3 and various temperatures prior to substrate addition on its activity towards D-Asp. The enzymatic activity is presented as a percentage relative to that in the absence of preincubation. (D) The effect of a 30 min preincubation of human DDO at 45°C and various pH levels prior to substrate addition on its activity towards D-Asp. The buffers used for each pH value are described in Materials and Methods. The enzymatic activity is presented as a percentage relative to that in the absence of preincubation. (A–D) Data are presented as the mean±standard deviation of three independent assays. Where not evident, the error bars are smaller than the symbols used.

Next, the effect of temperature on the stability of human DDO was examined. In these experiments, prior to the addition of the D-Asp substrate, human DDO was preincubated at temperatures ranging from 30°C to 70°C for 30 min at pH 8.3. There was no loss of activity until the preincubation temperature reached 45°C, and only 9% of the maximum activity was lost at 50°C (Fig. 1C). Human DDO retained half of its maximum activity at 55°C, while almost all of the activity was lost at 60°C. The effect of pH on the stability of human DDO was also examined by preincubating human DDO at pH levels ranging from 7.5 to 12.0 for 30 min at 45°C, prior to the addition of D-Asp. Human DDO was stable at pH 8.3 to 11.0 and retained 80% and 60% of its maximum activity at pH 7.5 and pH 12.0, respectively (Fig. 1D).

Substrate Specificity of Human DDO

The substrate specificity of human DDO was determined using various D-amino acids and their enantiomers as substrates (Table 1). Human DDO displayed similar enzymatic activities towards D-Asp and NMDA, but relatively low activities towards D-Glu and N-methyl-D-Glu. In addition to the acidic D-amino acids, D-Asn, D-Pro and D-His were also oxidized by human DDO; however, the enzymatic activities towards these D-amino acids were very low (3.4%, 0.14% and 0.17% of the activity towards D-Asp, respectively). With the exception of N-methyl-L-Asp, no activity towards any of the L-amino acids examined was detected. These results indicate that human DDO is highly specific to D-Asp and NMDA. This substrate specificity profile of human DDO is similar to the previously reported substrate preference of mouse DDO³²⁾; namely, mouse DDO is also highly specific to D-Asp and NMDA (Table 1). However, the relative levels of these DDO activities significantly differ from each other. In addition, mouse DDO showed no activity towards D-His and N-methyl-L-Asp, while human DDO displayed very low activity towards these amino acids. Thus, these findings suggest that the active site of human DDO is structurally different from that of the mouse DDO.

Table 1. Enzymatic Activities of Recombinant Human and Mouse DDOs Towards Various D- and L-Amino Acids

Substrate	Specific activity (µmol/min/mg)
	Human DDO a)	Mouse DDO b)
D-Asp	87.5±12.3	12.2±0.9
NMDA	91.2±14.8	13.1±0.6
D-Glu	7.82±1.39	0.46±0.07
N-Methyl-D-Glu	3.17±0.13	—c)
D-Asn	2.93±0.47	0.16±0.02
D-Pro	0.12±0.04	—
D-His	0.14±0.06	ND d)
N-Methyl-L-Asp	0.47±0.08	ND

a) Data are shown as the mean±standard deviation of three independent assays. Human DDO showed no activity against D-Gln, D-Ser, D-Thr, D-Tyr, D-Ala, D-Val, D-Leu, D-Ile, D-Phe, D-Met, D-Trp, D-cysteine, D-Lys, D-Arg, L-Asp, L-Glu, N-methyl-L-Glu, L-Asn, L-Gln, L-Ser, L-Thr, L-Tyr, L-Ala, L-Val, L-Leu, L-Ile, L-Pro, L-Phe, L-Met, L-Trp, L-cysteine, L-Lys, L-Arg, L-His, or Gly. b) Data are from Katane et al. (40 mM sodium pyrophosphate buffer, pH 8.3, 37°C).³²⁾ Mouse DDO showed no activity against D-Gln, D-Ser, D-Thr, D-Ala, D-Val, D-Leu, D-Phe, D-Met, D-Trp, D-cysteine, D-Lys, D-Arg, L-Asp, L-Glu, L-Asn, L-Gln, L-Ser, L-Thr, L-Ala, L-Val, L-Leu, L-Phe, L-Met, L-Trp, L-cysteine, L-Lys, L-Arg, or L-His.³²⁾ c) Not tested. d) Not detected.

The substrate specificity of human DDO differs markedly from that reported for non-mammalian DDO, such as DDOs of octopus, the nematode Caenorhabditis elegans, and the yeast Cryptococcus humicola. Specifically, the octopus DDO displays similar enzymatic activities towards D-Asp and D-Glu, but relatively low activity towards NMDA.⁴⁷⁾ The C. elegans contains genes encoding at least three functional DDOs (DDO-1, -2 and -3), and all the C. elegans DDOs display substantial activities towards D-Glu in addition to D-Asp and NMDA.³⁴⁾ The C. humicola DDO is highly specific to D-Asp, and the activities of this enzyme towards NMDA and D-Glu are remarkably lower than that towards D-Asp.⁴⁸⁾ Taken together, it appears that the active site structures of these non-mammalian DDOs are considerably different from one another and from those of mammalian DDOs, including the human enzyme. Indeed, it was reported recently that an active site residue Arg-243 of the C. humicola DDO is not important in its substrate specificity,⁴⁸⁾ while an active site residue Arg-216 of mouse DDO, which is structurally equivalent to Arg-243 of the C. humicola DDO, was shown to play crucial role(s) in its substrate specificity.³²⁾

Effects of Various Compounds on the Activity of Human DDO

Next, the effects of various compounds on the enzymatic activity of human DDO towards D-Asp were examined. With the exception of aminooxyacetic acid, which reduced the enzymatic activity of human DDO significantly, none of the other compounds tested affected DDO activity (Table 2). Aminooxyacetic acid is a representative inhibitor of pyridoxal 5′-phosphate-dependent enzymes; to determine the type of inhibition of human DDO by this compound, 1/v versus 1/[D-Asp] plots, which show the apparent kinetic parameters (K_m and V_max) for D-Asp, were generated in the absence or presence of aminooxyacetic acid. These plots revealed that aminooxyacetic acid inhibited the enzymatic activity of human DDO in a non-competitive manner (data not shown).

Table 2. The Effects of Various Compounds on the Enzymatic Activity of Recombinant Human DDO

Compound	Concentration (mM)	Relative activity (%)a)
None	—	100±5
EDTA	1.0	104±6
NaCl	1.0	98.2±5.2
KCl	1.0	98.9±5.9
MgCl2	1.0	96.8±4.6
FeCl2	1.0	86.5±5.2
CaCl2	1.0	100±9
CoCl2	1.0	101±9
CuCl2	1.0	90.7±8.1
NiCl2	1.0	93.3±13.5
MnCl2	1.0	100±4
ZnCl2	1.0	103±3
ATP	1.0	105±11
ADP	1.0	104±5
AMP	1.0	102±8
cAMP	1.0	102±7
Pyridoxal-5′-phosphate	0.2	93.5±5.3
Aminooxyacetic acid	1.0	66.4±4.0 b)
L-Asp	100	101±2
L-Glu	100	89.3±4.4

a) Data are shown as the mean±standard deviation of three independent assays. Enzymatic activity is presented as a percentage relative to the activity in the absence of compound. b) p<0.001 (Dunnett’s multiple comparison test) compared to the activity in the absence of compound.

The addition of L-Asp or L-Glu to the reaction mixture had no significant effect on the enzymatic activity of human DDO (Table 2), which is consistent with the previous report of a lack of inhibition of rabbit DDO activity by these L-amino acids.²⁹⁾ Other than the aforementioned study, to our knowledge, there have been no previous reports of the effects of the compounds listed in Table 2 on the activity of mammalian DDO. On the other hand, it was reported recently that the activity of DDO from the fungus Trichoderma harzianum is inhibited by several metals, including MgCl₂, NiCl₂ and CuCl₂ (43%, 94% and 99% inhibition, respectively)⁴⁹⁾; however, these metals had no significant effect on the activity of human DDO (Table 2). Although relatively higher concentrations of these metals were used in the study of T. harzianum DDO (5 mM compared with 1 mM in this study), the susceptibilities of the human and T. harzianum enzymes to these metals appear to differ strikingly.

Characterization and Comparison of the Kinetic Properties of Human and Rodent DDOs

The apparent kinetic parameters (K_m and k_cat) of human, rat and mouse DDOs were determined using D-Asp as a substrate. The rat and mouse proteins were expressed in E. coli and purified to near homogeneity, as described in Materials and Methods. The K_m value of human DDO towards D-Asp was comparable to that of rat DDO and was significantly lower than that of mouse DDO (Table 3). The k_cat value of human DDO towards D-Asp was significantly higher than those of the rat and mouse DDOs; therefore, the catalytic efficiency (k_cat/K_m) of human DDO was significantly higher than those of the rat and mouse enzymes. These parameters of the human and mouse DDOs are comparable to previously reported values.^32,34–36) These results suggest that the active site of human DDO is structurally different from those of the rat and mouse DDOs. Furthermore, the catalytic efficiency of human DDO towards D-Asp was higher than those reported for other mammalian (cow and pig) DDOs^46,50) (Table 3). Taken together, these findings suggesting that the active site of human DDO is more suitable for catalyzing the oxidative deamination of D-Asp than those of other mammalian DDOs.

Table 3. The Apparent Steady-State Kinetic Parameters of Recombinant Mammalian DDOs Using D-Asp as a Substrate

Enzyme	Km (mM)	kcat (s−1)	kcat/Km (M−1 s−1)
Human DDOa)	2.10±0.15	68.4±1.8	32651±1999
Rat DDOa)	2.26±0.44	31.1±2.3	14104±2447
Mouse DDOa)	7.37±0.87	9.83±0.51	1354±229
Cow DDOb)	3.7	22.5	6090
Pig DDOc)	2.52	37.5	14881

a) Data are shown as the mean±standard deviation of three independent assays. b) Data are from Negri et al. (50 mM sodium phosphate buffer, pH 7.4, 25°C).⁵⁰⁾ c) Data are from Yamamoto et al. (50 mM sodium pyrophosphate buffer, pH 8.3, 37°C).⁴⁶⁾

Characterization and Comparison of the Inhibitor Binding Properties of Human and Rodent DDOs

To investigate possible structural differences between the human, rat and mouse DDOs, binding of these enzymes to malonate and meso-tartrate was examined; these compounds reportedly inhibit mammalian DDO by competing with their substrates.^28,29,31) In addition, because aminooxyacetic acid was found to non-competitively inhibit DDO activity, binding of DDO to this compound was also examined. Malonate, meso-tartrate and aminooxyacetic acid all inhibited the enzymatic activities of human, rat and mouse DDOs towards D-Asp in a dose-dependent manner, and the IC₅₀ values of these compounds were determined (data not shown). Using the IC₅₀ values and the determined K_m values towards D-Asp, the K_i values of these compounds for human, rat and mouse DDOs were calculated^38,39) (Table 4). The lowest K_i value of malonate was obtained for human DDO, followed by mouse DDO and then rat DDO. The differences between these K_i values were significant (p<0.05 for rat versus mouse, and p<0.001 for human versus rat and human versus mouse, based on a Tukey–Kramer multiple comparison test). The K_i values of meso-tartrate for human and rat DDO were comparable (p>0.05; Tukey–Kramer test), and were significantly lower than that for mouse DDO (p<0.001; Tukey–Kramer test). In contrast to those of malonate and meso-tartrate, the K_i values of aminooxyacetic acid for the human, rat and mouse DDOs were comparable (p>0.05, Tukey–Kramer test). Taken together, these results provide further evidence that the active site of human DDO is structurally different from those of the rat and mouse DDOs.

Table 4. The K_i Values of Several Inhibitors against Recombinant Human, Rat and Mouse DDO

Inhibitor	Ki (µM)a)
	Human DDO	Rat DDO	Mouse DDO
Malonate	153±26	1562±120	1220±133
meso-Tartrate	681±48	472±87	2089±216
Aminooxyacetic acid	1492±693	1416±356	1915±100

a) Data are shown as the mean±standard deviation of three to four independent assays.

Insight into Differences between the Structures of Human and Rodent DDOs

The human, rat and mouse DDO/Ddo cDNAs all encode a protein of 341 amino acids. The amino acid sequence of human DDO shares 81.8% and 80.4% identity with that of rat and mouse DDO, respectively, and the rat and mouse enzymes share 91.2% identity. Among the 75 residues that differ between human DDO and its rodent counterparts, 45, 11, and 13 residues are present specifically in human, rat, and mouse DDO, respectively, and six residues differ in all three enzymes.

To gain further insights into the structural differences between the human and rodent DDOs, 3D structural models of the enzymes and a theoretical model of the human DDO/malonate complex were constructed in silico. Five regions that differed in their overall backbone structures among the three DDOs were identified (Fig. 2A). In region 1, the structure is different in mouse only; in regions 2 and 3, the structures are different in all three enzymes; and in regions 4 and 5, the structures are different in human only. Regions 2 and 5 participate in the formation of tunnel structure through which malonate presumably reaches the active site pocket (Fig. 2B); hence, these two regions may be involved in the migration of the substrate/product into and out of the active site. These findings suggest that the structural differences in regions 2 and 5 contribute, at least in part, to the distinctive kinetic and inhibitor binding properties of human DDO described above. On the other hand, a large number of the active site residues are well-conserved in the human, rat and mouse DDOs. However, it should be noted that the His-54 residue of human DDO is replaced by Pro in rat DDO (Fig. 2C), while it is conserved in mouse DDO. Additional studies are required to clarify the role of the His-54 residue in the enzymatic and structural properties of human DDO.

Fig. 2. Predicted 3D Structural Models of Human, Rat and Mouse DDO

(A) The proposed structural model of human DDO (green) is superimposed on those of rat DDO (magenta) and mouse DDO (orange). Malonate, which was docked at the active site of human DDO by a simulated annealing docking analysis (see main text for details), and FAD molecules are also shown. The five regions (1–5) that differ in their overall backbone structures between the three DDOs are indicated by blue arrows. (B) Surface representation of the theoretical model of the human DDO/malonate complex. The amino acids in regions 2 and 5 are highlighted in red. (C) The side-chains of the active site residues of human DDO. Pro-54 of rat DDO is structurally equivalent to His-54 of human DDO. The carbon atoms in the side-chains of amino acids of human and rat DDOs are colored green and purple, respectively.

CONCLUSION

The results presented here reveal the enzymatic properties of human DDO in detail. The optimum pH and temperature of human DDO were pH 8.3 to 12.5 and 45°C, respectively, and the enzyme was relatively stable until the temperature reached 45°C and the pH exceeded 11.0 (Fig. 1). Furthermore, human DDO was highly specific for D-Asp and NMDA (Table 1), and its enzymatic activity towards D-Asp was more efficient than those of the rat and mouse DDOs (Table 3). With the exception of aminooxyacetic acid, the enzymatic activity of human DDO was not affected by the presence of a number of different compounds (Table 2). This study also identified differences between the predicted 3D structures of the human and rodent DDOs (Fig. 2). Notably, the differences between these proteins occurred in regions that appear to be involved in migration of the substrate/product into and out of the active site. These structural differences may be reflected by the distinct kinetic and inhibitor binding properties of human DDO compared with its rat and mouse homologs (Tables 3, 4).

Preclinical studies using experimental rodents, such as rats and mice, are effective methods of evaluating the in vivo effects of DDO inhibitors as lead compounds for the development of new drugs; however, species-related differences in the properties of human and rodent DDOs may affect these studies and impact the development of effective inhibitors. From a pharmacological point of view, consideration of the distinct properties of human DDO in relation to those of its rodent counterparts is required to ensure accurate evaluation of the in vivo effects of lead compounds.

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research (21590090) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by a Kitasato University Research Grant for Young Researchers (to M.K.).

Conflict of Interest

The authors declare no conflict of interest.

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