Amino Acids

The extraction and comparison of soil bio-available amino acids using either demineralised water (DEMI H(2)O) or 0.5 M ammonium acetate (0.5 M AAc) solution is reported. Results show that the extraction by 0.5 M AAc is a better method to assess the concentration of bio-available amino acids in soil than DEMI H(2)O due to higher extraction efficiency and better amino acid protection against microbial degradation during processing.

Recently, Guo et al. have reported structural as well as the binding energy data of the particular interactions between the cleavage sites of hemagglutinin and serine proteases, trypsin and furin, using molecular docking approach. Due to a wrong assignment of protonation state on the histidine, one of the catalytic triad in the active site of both enzymes, their docking results are contradictory with the fundamental principle and previous theoretical studies of the known cleavage mechanism in serine proteases.

Ionic interactions are essential for the biological functions of the polyamines spermidine and spermine in mammalian physiology. Here, we describe a simple gram scale method to prepare 1,12-diamino-3,6,9-triazadodecane (SpmTrien), an isosteric charge-deficient spermine analogue. The protonation sites of SpmTrien were determined at pH range of 2.2–11.0 using two-dimensional 1H-15N NMR spectroscopy, which proved to be more feasible than conventional methods. The macroscopic pK a values of SpmTrien (3.3, 6.3, 8.5, 9.5 and 10.3) are significantly lower than those of 1,12-diamino-4,9-diazadodecane (spermine). The effects of SpmTrien and its parent molecule, 1,8-diamino-3,6-diazaoctane (Trien), on cell growth and polyamine metabolism were investigated in DU145 prostate carcinoma cells. SpmTrien downregulated the biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosyl-l-methionine decarboxylase and decreased intracellular polyamine levels, whereas the effects of Trien alone were minor. Interestingly, both SpmTrien and Trien were able to partially overcome growth arrest induced by an ODC inhibitor, α-difluoromethylornithine, indicating that they are able to mimic some functions of the natural polyamines. Thus, SpmTrien is a novel tool to influence polyamine interaction sites at the molecular level and offers a new means to study the contribution of the protonation of spermine amino group(s) in the regulation of polyamine-dependent biological processes.

Taurine, a known antioxidant and neuroprotector has been investigated for its free radical scavenging action in vitro in isolated mitochondria, and tested whether it protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration in mice. Taurine (0.1-10 mM) did not affect 1-methyl-4-phenyl pyridinium-induced hydroxyl radical production in isolated mitochondria. Systemic administration of taurine (250 mg/kg, i.p.) caused a small, but significant loss of dopamine levels in the striatum of mice. Taurine failed to reverse MPTP-induced striatal dopamine depletion, but caused significant increase in dopamine turnover in these animals. In the light of the present study it may be suggested that consumption of taurine may neither help in scavenging of neurotoxic hydroxyl radicals in the brain mitochondria, nor would it help in blocking the process of neurodegeneration.

Ghrelin is a 28-residue peptide acylated with an n-octanoyl group on the Ser 3 residue, predominantly produced by the stomach. Ghrelin displays strong growth hormone (GH) releasing activity, which is mediated by the activation of the so-called GH secretagogue receptor type 1a (GHS-R1a). Given the wide spectrum of biological activities of Ghrelin in neuroendocrine and metabolic pathways, many research groups, including our group, developed synthetic peptide, and nonpeptide GHS-R1a ligands, acting as agonists, partial agonists, antagonists, or inverse agonists. In this highlight article, we will focus on the discovery of a GHS-R1a antagonist compound, JMV 2959, which has been extensively studied in different in vitro and in vivo models. We will first describe the peptidomimetic approach that led us to discover this compound. Then we will review the results obtained with this compound in different studies in the fields of food intake and obesity, addictive behaviors, hyperactivity and retinopathy.

Constrained enantiopure bicyclic β-amino acids derived from the asymmetric Diels-Alder reaction of the (R)-benzyl-4-(3-acryloyloxy-4,4-dimethyl-2-oxopyrrolidin-1-yl)-benzoate and the 1-(benzyloxycarbonylamino)cyclohexadiene provide original templates for the construction of new rigid enantiopure 1,3-amino alcohols.

This work deals with the Dakin-West synthesis, starting from the nucleoamino acid 1-thyminyl acetic acid, as well the NMR, ESI MS, and X-ray characterization of a heteroaromatic compound denominated by us T(2)CO, comprising two thymine moieties anchored to a 2-propanonic unit, the spectroscopic properties of which were studied by UV as a function of temperature and ionic strength. Preliminary binding-studies with molecules of biomedical interest such as nucleic acids and proteins, performed on samples containing T(2)CO, suggested that this molecule is able to interact very weakly with double-stranded RNA, whereas it does not seem to bind other nucleic acids or proteins. Moreover, by studies with fresh human serum we found that T(2)CO is resistant to enzymatic degradation till 24 h, whereas UV metal binding-studies, performed using solutions of copper (II) chloride dihydrate and nickel (II) chloride hexahydrate, revealed a certain ability of T(2)CO to bind copper (II) cation. Finally, by CD spectroscopy we investigated the influence of T(2)CO on the already described supramolecular networks based on L-serine-containing nucleopeptides. More particularly, we found that T(2)CO is able to increase the level of structuration of the non-covalent supramolecular assembly of the chiral nucleopeptides, which is a feature of remarkable interest for the development of innovative drug delivery tools.

Highly emissive heterocyclic asparagine derivatives bearing a 1,3,4-thiadiazolyl unit at the side chain, functionalised with electron donor or acceptor groups, were synthesised and evaluated as amino acid-based fluorimetric chemosensors for metal cations, such as Cu²(+), Zn²(+), Co²(+) and Ni²(+). The results suggest that there is a strong interaction through the donor heteroatoms at the side chain of the various asparagine derivatives, with high sensitivity towards Cu²(+) in a ligand-metal complex with 1:2 stoichiometry. Association constants and detection limits for Cu²(+) were calculated. The photophysical and metal ion sensing properties of these asparagine derivatives confirm their potential as fluorimetric chemosensors and suggest that they can be suitable for incorporation into chemosensory peptidic frameworks.

(S)- and (R)-BIMBOL were efficient PT catalysts of asymmetric Michael addition of prochiral Ni-PBP-Gly (1) to acrylic esters and malonic esters to Ni-PBP-Δ-Ala (2) correspondingly. The salient feature of the catalysis is opposite configurations of Glu prepared via the two paths with BIMBOL of the same configuration and a perspective novel catalytic procedure for the synthesis of Gla derivatives.

Antimicrobial peptides (AMPs) are a promising solution to face the antibiotic-resistant problem because they display little or no resistance effects. Dimeric analogues of select AMPs have shown pharmacotechnical advantages, making these molecules promising candidates for the development of novel antibiotic agents. Here, we evaluate the effects of dimerization on the structure and biological activity of the AMP aurein 1.2 (AU). AU and the C- and N-terminal dimers, (AU)2K and E(AU)2, respectively, were synthesized by solid-phase peptide synthesis. Circular dichroism spectra indicated that E(AU)2 has a "coiled coil" structure in water while (AU)2K has an α-helix structure. In contrast, AU displayed typical spectra for disordered structures. In LPC micelles, all peptides acquired a high amount of α-helix structure. Hemolytic and vesicle permeabilization assays showed that AU has a concentration dependence activity, while this effect was less pronounced for dimeric versions, suggesting that dimerization may change the mechanism of action of AU. Notably, the antimicrobial activity against bacteria and yeast decreased with dimerization. However, dimeric peptides promoted the aggregation of C. albicans. The ability to aggregate yeast cells makes dimeric versions of AU attractive candidates to inhibit the adhesion of C. albicans to biological targets and medical devices, preventing disease caused by this fungus.

Site-directed mutagenesis study was performed to elucidate the role of conserved tryptophan-101 present at the active site of phosphoserine aminotransferase from an enteric human parasite Entamoeba histolytica. Fluorescence resonance energy transfer and molecular dynamic simulation show that the indole ring of Trp101 stacks with the cofactor PLP. Loss of enzymatic activity and PLP polarization values suggest that Trp101 plays a major role in maintaining a defined PLP microenvironment essentially required for optimal enzymatic activity. Studies on W101F, W101H and W101A mutants show that only the indole ring of the conserved Trp101 forms most favorable stacking interaction with the pyridine ring of the cofactor PLP. Protein stability was compromised on substitution of Trp101 with Phe/His/Ala amino acids. A difference in conformational free energy of 1.65 kcal mol−1 was observed between WT-protein and W101A mutant.

We have previously reported that the proline-rich decapeptide from Bothrops jararaca (Bj-PRO-10c) causes potent and sustained antihypertensive and bradycardic effects in SHR. These activities are independent of ACE inhibition. In the present study, we used the Ala-scan approach to evaluate the importance of each amino acid within the sequence of Bj-PRO-10c (Pyr(1)-Asn(2)-Trp(3)-Pro(4)-His(5)-Pro(6)-Gln(7)-Ile(8)-Pro(9)-Pro(10)). The antihypertensive and bradycardic effects of the analogues Bj-PRO-10c Ala(3), Bj-PRO-10c Ala(7), Bj-PRO-10c Ala(8) were similar to those of Bj-PRO-10c, whereas the analogues Bj-PRO-10c Ala(2), Bj-PRO-10c Ala(4), Bj-PRO-10c Ala(5), Bj-PRO-10c Ala(9), and Bj-PRO-10c Ala(10) kept the antihypertensive activity and lost bradycardic activity considerably. In contrast, Bj-PRO-10c Ala(1) and Bj-PRO-10c Ala(6) were unable to provoke any cardiovascular activity. In summary, we demonstrated that (1) the Pyr(1) and Pro(6) residues are essential for both, the antihypertensive and bradycardic effects of Bj-PRO-10c; (2) Ala-scan approach allowed dissociating blood pressure reduction and bradycardic effects. Conformational properties of the peptides were examined by means of circular dichroism (CD) spectroscopy. The different Ala-scan analogues caused either an increase or decrease in the type II polyproline helix content compared to Bj-PRO-10c. The complete loss of activity of the Pro(6) → Ala(6) mutant is probably due to the fact that in the parent peptide the His(5)-Pro(6) bond can exist in the cis configuration, which could correspond to the conformation of this bond in the bound state. Current data support the Bj-PRO-10c as a promising leader prototype to develop new agents to treat cardiovascular diseases and its co-morbidities.

A series of PTH hybrids containing a diamine [NH2(CH2)n NH2; n = 4, 5, 6] in the C-terminal position was synthesized based on the H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2 (Har = homoarginine) template. The compounds were pharmacologically characterized at PTH1R receptors for agonist activity.

Ornithine decarboxylase antizyme 1 (AZ1) is a major regulatory protein responsible for the regulation and degradation of ornithine decarboxylase (ODC). To better understand the role of AZ1 in polyamine metabolism and in modulating the response to anticancer polyamine analogues, a small interfering RNA strategy was used to create a series of stable clones in human H157 non-small cell lung cancer cells that expressed less than 5-10% of basal AZ1 levels. Antizyme 1 knockdown clones accumulated greater amounts of the polyamine analogue N (1),N (11)-bis(ethyl)norspermine (BENSpm) and were more sensitive to analogue treatment. The possibility of a loss of polyamine uptake regulation in the knockdown clones was confirmed by polyamine uptake analysis. These results are consistent with the hypothesis that AZ1 knockdown leads to dysregulation of polyamine uptake, resulting in increased analogue accumulation and toxicity. Importantly, there appears to be little difference between AZ1 knockdown cells and cells with normal levels of AZ1 with respect to ODC regulation, suggesting that another regulatory protein, potentially AZ2, compensates for the loss of AZ1. The results of these studies are important for the understanding of both the regulation of polyamine homeostasis and in understanding the factors that regulate tumor cell sensitivity to the anti-tumor polyamine analogues.

Depletion of pancreatic intracellular polyamine pools has been observed in acute pancreatitis both in the animal models and in humans. In this study, the wild-type mice, polyamine catabolic enzyme spermidine/spermine N 1-acetyltransferase overexpressing (SSAT mice) and SSAT-deficient mice were used to characterize the new zinc-induced acute pancreatitis mouse model and study the role of polyamines and polyamine catabolism in this model. Intraperitoneal zinc injection induced acute necrotizing pancreatitis in wild-type mice as well as in SSAT-overexpressing and SSAT-deficient mice. Serum α-amylase activity was significantly increased in all zinc-treated mice compared with the untreated controls. However, the α-amylase activities in SSAT mice were constantly lower than those in the other groups. Histopathological examination of pancreatic tissue revealed edema, acinar cell necrosis and necrotizing inflammation, typical for acute pancreatitis. Compared with the other zinc-treated mice less damage according to the histopathological analysis was observed in the pancreatic tissue of SSAT mice. Levels of intracellular spermidine, and occasionally spermine, were significantly decreased in pancreases of all zinc-treated animals and SSAT enzyme activity was enhanced both in wild-type and SSAT mice. Interestingly, a spermine analog, N 1, N 11-diethylnorspermine (DENSpm), enhanced the proliferation of pancreatic cells and reduced the severity of zinc-induced pancreatitis in wild-type mice. The results show that in mice a single intraperitoneal zinc injection causes acute necrotizing pancreatitis accompanied by decrease of intracellular polyamine pools. The study supports the important role of polyamines for the integrity and function of the pancreas. In addition, the study suggests that whole body overexpression of SSAT obtained in SSAT mice reduces inflammatory pancreatic cell injury.

Carbon-11 (β+ emitter, t 1/2 = 20.4 min) radiolabeled l-glutamine is a potentially useful molecular imaging agent that can be utilized with positron emission tomography for both human oncological diagnosis and plant imaging research. Based upon a previously reported [11C]cyanide end-capping labeling method, a systematic investigation of nucleophilic cyanation reactions and acidic hydrolysis reaction parameters, including base, metal ion source, phase transfer catalyst, solvent, reaction temperature and reaction time, was conducted. The result was a milder, more reliable, two-step method which provides l-[5-11C]-glutamine with a radiochemical yield of 63.8 ± 8.7 % (range from 51 to 74 %, n = 10) with >90 % radiochemical purity and >90 % enantiomeric purity. The total synthesis time was 40–50 min from the end of bombardment. In addition, an Fmoc derivatization method was developed to measure the specific activity of this radiotracer.

Maternal protein restriction diminishes placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) activity and causes fetal growth restriction in mammals. However, it is unknown whether such effect was caused directly by nutrient deficiency, or indirectly through the mediation of maternal hormones. In the present study, a human placental cell line (BeWo) was cultured in F12K as control and F12 as low amino acids (LAA) media for 48 h to investigate the effects of amino acids deficiency on 11β-HSD2 expression and activity. Despite a significant up-regulation of 11β-HSD2 mRNA expression in LAA cells, 11β-HSD2 activity and protein content were decreased by 38 and 54%, respectively (P<0.05), indicating a mechanism of post-transcriptional regulation. Among 5 miRNAs targeting 11β-HSD2, miR-498 was expressed significantly higher in LAA cells. Leptin concentration was significantly lower (P<0.01) in LAA medium. The mRNA expression of both isoforms of leptin receptor was significantly higher in LAA cells, although no difference was detected at protein level. To further clarify whether leptin is involved in mediating the effect of LAA on 11β-HSD2 activity, leptin was supplemented to LAA medium, whereas three specific inhibitors of leptin signaling pathways, WP1066 for JAK-STAT, PD98059 for MAPK and LY294002 for PI3K, respectively were added to control medium. Leptin restored the diminished 11β-HSD2 activity in LAA cells, whereas WP1066 (5 nM) and PD98059 (50 nM) significantly decreased 11β-HSD2 activity in control cells. In conclusion, the present results indicate that LAA diminishes 11β-HSD2 expression and activity in BeWo cells through leptin-activated JAK-STAT and MAPK pathways.

S-(11)C-methyl-L-cysteine (LMCYS) is an attractive amino acid tracer for clinical tumor positron emission tomography (PET) imaging. D-isomers of some radiolabeled amino acids are potential PET tracers for tumor imaging. In this work, S-(11)C-methyl-D-cysteine (DMCYS), a D-amino acid isomer of S-(11)C-methyl-cysteine for tumor imaging was developed and evaluated. DMCYS was prepared by (11)C-methylation of the precursor D-cysteine, with an uncorrected radiochemical yield over 50 % from (11)CH3I within a total synthesis time from (11)CO2 about 12 min. In vitro competitive inhibition studies showed that DMCYS uptake was primarily transported through the Na(+)-independent system L, and also the Na(+)-dependent system B(0,+) and system ASC, with almost no system A. In vitro incorporation experiments indicated that almost no protein incorporation was found in Hepa 1-6 hepatoma cell lines. Biodistribution studies demonstrated higher uptake of DMCYS in pancreas and liver at 5 min post-injection, relatively lower uptake in brain and muscle, and faster radioactivity clearance from most tissues than those of L-isomer during the entire observation time. In the PET imaging of S180 fibrosarcoma-bearing mice and turpentine-induced inflammatory model mice, 2-(18)F-fluoro-2-deoxy-D-glucose (FDG) exhibited significantly high accumulation in both tumor and inflammatory lesion with low tumor-to-inflammation ratio of 1.40, and LMCYS showed low tumor-to-inflammation ratio of 1.64 at 60 min post-injection. By contrast, DMCYS showed moderate accumulation in tumor and very low uptake in inflammatory lesion, leading to relatively higher tumor-to-inflammation ratio of 2.25 than (11)C-methyl-L-methionine (MET) (1.85) at 60 min post-injection. Also, PET images of orthotopic transplanted glioma models demonstrated that low uptake of DMCYS in normal brain tissue and high uptake in brain glioma tissue were observed. The results suggest that DMCYS is a little better than the corresponding L-isomers as a potential PET tumor-detecting agent and is superior to MET and FDG in the differentiation of tumor from inflammation.

The N-terminal 1-34 fragment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses characteristic of the native intact PTH. Recently, analogues of PTH(1-11) fragments with helicity-enhancing substitutions have been demonstrated to yield potent analogues of PTH(1-34). The work describes the synthesis, biological activity and structure of analogues of the best modified PTH sequence H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2 (I). In particular, the effect of the Ala/Aib substitution at positions 1 and 3 as well as of the replacement of Nle in position 8 with D-Nle, L-(αMe)-Nle and D-(αMe)-Nle was studied. The resulting peptides were characterized structurally by CD spectroscopy, solution NMR and MD, and in vitro for activity with respect to the cognate receptor, parathyroid hormone receptor.

The N-terminal 1-34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it elicits all the biological responses characteristic of the native intact PTH. Recent studies reported potent helical analogues of the PTH (1-11) with helicity-enhancing substitutions. This work describes the synthesis, biological activity, and conformational studies of analogues obtained from the most active non-natural PTH (1-11) peptide H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2; specifically, the replacement of Val in position 2 with D-Val, L-(αMe)-Val and N-isopropyl-Gly was studied. The synthesized analogues were characterized functionally by in-cell assays and their structures were determined by CD and NMR spectroscopy. To clarify the relationship between the structure and activity, the structural data were used to generate a pharmacophoric model, obtained overlapping all the analogues. This model underlines the fundamental functional role of the side chain of Val2 and, at the same time, reveals that the introduction of conformationally constrained Cα-tetrasubstituted α-amino acids in the peptides increases their helical content, but does not necessarily ensure significant biological activity.

A fast and efficient microwave-assisted solid phase peptide synthesis (MW-SPPS) of a 51mer peptide, the main heparin-binding site (60-110) of human pleiotrophin (hPTN), using 2-chlorotrityl chloride resin (CLTR-Cl) following the 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) methodology and with the standard N,N'-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) coupling reagents, is described. An MW-SPPS protocol was for the first time successfully applied to the acid labile CLTR-Cl for the faster synthesis of long peptides (51mer peptide) and with an enhanced purity in comparison to conventional SPPS protocols. The synthesis of such long peptides is not trivial and it is generally achieved by recombinant techniques. The desired linear peptide was obtained in only 30 h of total processing time and in 51% crude yield, in which 60% was the purified product obtained with 99.4% purity. The synthesized peptide was purified by reversed phase high performance liquid chromatography (RP-HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS). Then, the regioselective formation of the two disulfide bridges of hPTN 60-110 was successfully achieved by a two-step procedure, involving an oxidative folding step in dimethylsulfoxide (DMSO) to form the Cys(77)-Cys(109) bond, followed by iodine oxidation to form the Cys(67)-Cys(99) bond.

Peptide 11389 from CD21-binding region of EBV-gp350/220 protein binds to PBMCs inducing IL-6 expression and inhibiting EBV-binding to PBMCs. In addition, anti-peptide 11389 antibodies recognize EBV-infected cells and inhibit both EBV infection and IL-6 production in PBMCs. We have postulated that native structure stabilization of peptide 11389 sequence can increase its biological activity. The strategy was to modify its sequence to restrict the number of structures that peptide 11389 could acquire in solution (decreasing peptide's configurational entropy) and to weaken the non-relevant intermolecular interactions (decreasing its hydrophobicity), preserving CD21-interacting residues and structure as displayed in the native protein. Thirteen analog peptides were designed and synthesized; most of them were monomers containing an intra-chain disulfide bridge. Analog peptides 34058, 34060, 34061, 34296, 34298, 34299 and 34300 inhibited EBV invasion of PBMCs. Peptides 34059, 34060, 34295 and 34297 induced IL-6 levels in PBMCs (EC50=3.4, 3.3, 0.5, 0.5 μM, respectively) at higher potency than peptide 11389 (EC50=5.8 μM). Peptides 34057, 34059, 34060, 34301 and 34302 interacted with anti-EBV antibodies with affinities from 3 to 50 times higher than peptide 11389. Most of analog peptides were highly immunogenic and elicited antibodies that cross-react with EBV. In conclusion, we have designed peptides displaying higher biological activity than peptide 11389.

No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.

Handling and detoxification of metals by enzymes is a major issue that is not in the focus of current biomedical research concepts. The finding of the presence of arsenic (+3 Oxidation State) methyltransferase (AS3MT) in neuroblastoma cells NE-115 as a high abundance protein made us investigate primary structure of AS3MT reflecting an example of metal-handling in eucaryotes. Proteins extracted from NE-115 cells were run on 2-DE followed by two different mass spectrometrical methods. High sequence coverage was obtained by multiple protease digestion and a sequence conflict was solved at arginine 335. These findings are important when future studies on this enzyme are designed at the protein level and in particular, when antibodies against this protein will be generated.

Helicobacter pylori is a microaerophilic bacterium, associated with gastric inflammation and peptic ulcers. d-Amino acid dehydrogenase is a flavoenzyme that digests free neutral d-amino acids yielding corresponding 2-oxo acids and hydrogen. We sequenced the H. pylori NCTC 11637 d-amino acid dehydrogenase gene, dadA. The primary structure deduced from the gene showed low similarity with other bacterial d-amino acid dehydrogenases. We purified the enzyme to homogeneity from recombinant Escherichia coli cells by cloning dadA. The recombinant protein, DadA, with 44 kDa molecular mass, possessed FAD as cofactor, and showed the highest activity to d-proline. The enzyme mediated electron transport from d-proline to coenzyme Q1, thus distinguishing it from d-amino acid oxidase. The apparent K m and V max values were 40.2 mM and 25.0 μmol min−1 mg−1, respectively, for dehydrogenation of d-proline, and were 8.2 μM and 12.3 μmol min−1 mg−1, respectively, for reduction of Q1. The respective pH and temperature optima were 8.0 and 37°C. Enzyme activity was inhibited markedly by benzoate, and moderately by SH reagents. DadA showed more similarity with mammalian d-amino acid oxidase than other bacterial d-amino acid dehydrogenases in some enzymatic characteristics. Electron transport from d-proline to a c-type cytochrome was suggested spectrophotometrically.