A biosensor for all D-amino acids using evolved D-amino acid oxidase

Determination of the D-amino acid content in foods and in biological samples is a very important task. In order to achieve this goal we developed a biosensor employing the flavoenzyme D-amino acid oxidase from the yeast Rhodotorula gracilis. To produce a device in which the D-amino acid composition does not alter the results, both the wild-type and a number of mutants obtained by rational design and directed evolution approaches were used. An analysis of D-amino acid oxidase mutants activity on D-amino acid mixtures containing various ratios of neutral, acidic, and basic substrates identified the Amberzyme-immobilized T60A/Q144R/K152E and M213G mutants as the best choice: their response shows an only limited dependence on the solution composition when at least 20% of the D-amino acid is made up of D-alanine (standard error is approximately 5-9%). This is the first report, to our knowledge, demonstrating that the entire D-amino acid content can be determined by using a screen-printed electrode amperometric biosensor, with a detection limit of 0.25 mM and a mean response time of 10-15 min. The D-amino acid assay based on R. gracilis DAAO-biosensor is inexpensive, simple to perform, and rapid: the D-amino acid concentration of a variety of biological samples can be investigated using this assay.     

Determination of free D-proline and D-leucine in the brains of mutant mice lacking D-amino acid oxidase activity.

A new procedure to accurately measure a trace amount of d-proline in biological samples has been developed. This D-amino acid was derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and was determined by a column-switching HPLC system, a combination of a micro-ODS column and a chiral column. The detection limit for D-proline spiked in a mouse cerebrum sample is 1 fmol (injection amount, S/N = 3). Within-day precision and day-to-day precision obtained for spiked d-proline (10 fmol) are 2.14 and 5.35% (RSD), respectively. Using the new method, the amount of free D-proline in eight brain regions and sera of mutant ddY/DAO- mice, lacking D-amino acid oxidase activity, and control ddY/DAO+ mice was determined. The amount of free D-leucine was also investigated. The amount and distribution of D-proline in the brains of ddY/DAO+ mice and ddY/DAO- mice are almost the same, and relatively high amounts of D-proline have been observed in the pituitary gland and in the pineal gland. On the other hand, the amount of D-leucine is different between the two strains. In the brains of ddY/DAO+ mice, a relatively high amount of D-leucine has been observed in the pineal gland compared with other regions. In the brains of ddY/DAO- mice, D-leucine amounts are approximately 10 times higher than those obtained in ddY/DAO+ mice and regional difference has not been observed, while the amounts of L-proline and L-leucine are not significantly different between the two strains. In the serum, the amounts of both free D-proline and d-leucine are significantly higher in the ddY/DAO- mice than those obtained in ddY/DAO+ mice.     

Determination of D-amino acids using a D-amino acid oxidase biosensor with spectrophotometric and potentiometric detection

An amperometric and a colorimetric biosensor to detect and quantify D-amino acids selectively has been devised using D-amino acid oxidase from Rhodotorula gracilis. The sensor is characterised by a proportional response between 0.2-3 mM and 0.1-1 mM D-alanine for the amperometric (at a working potential of 1400 mV vs Ag/AgCl) and colorimetric system, respectively.     

Crystallographic studies on D-amino acid oxidase.

D-amino acid oxidase, a flavoprotein from hog kidneys, has been crystalized in two different forms. Orthorhombic prisms have been obtained from the enzyme.benzoate complex at pH 8.3; the space group is C2221 and the cell dimensions are a = 325A, b = 138.8 A, c = 200 A. At lower pH values, the enzyme crystallizes in trigonal prisms with a = b = 116.0 A, c = 399 A, space group P3112 or its enantiomorph. The two crystal forms have been obtained at 28 degrees C while at 4 degrees C only weak evidence of crystallization has been detected. In both crystalline modifications, the protein is highly associated.     

D-amino acid oxidase in mouse liver--II

1. Activity of D-amino acid oxidase was detected in tissue extract of mouse liver by two sensitive spectrophotometric methods. 2. The activity was also detectable in extracts of the heart, but not of lung.     

Immobilization and characterization of D-amino acid oxidase.

Optimal conditions with respect to pH, concentration of glutaraldehyde and enzyme, and order of addition of enzyme and crosslinking reagent were established for the immobilization of hog kidney D-amino acid oxidase to an attapulgite support. Yields of 40 to 70% were generally attained although when low concentrations of enzyme were used yields were consistently greater than 100%. It is suggested that this is due to a dimer leads to monomer shift at low protein concentrations. The stability of soluble D-amino acid oxidase was dependent on the buffer in which it was stored (pyrophosphate-phosphate greater than borate greater than Tris). Stability of immobilized enzyme was less than soluble in pyrophosphate-phosphate buffer, but storage in the presence of FAD improved stability. In addition, treatment of stored, immobilized enzyme with FAD before assay restored some of its activity. The immobilized D-amino acid oxidase was less stable to heat (50 degrees C) than the soluble enzyme from pH 6 to 8 but was more stable above and below these values. Apparent Km values for D-alanine, D-valine, and D-tryptophan decreased for the immobilized enzyme compared to the soluble.     

Purification of Rhodotorula gracilis D-amino acid oxidase.

A protocol is presented for preparing Rhodotorula gracilis D-amino acid oxidase in homogeneous form and in high yield in 3 to 4 days. The method takes advantage of (a) cell rupture by alternate freeze-thawing, (b) use of DEAE-Sepharose to bind contaminants, and (c) enzyme binding to a Mono S column. The D-amino acid oxidase isolated by this means has the same spectral and catalytic properties as the enzyme previously obtained, and possesses improved long-term stability.     

Interaction of steroids with D-amino acid oxidase.

1. Progesterone inhibited D-amino acid oxidase (D-amino acid : O2 oxidoreductase (deaminating), EC 1.4.3.3) in competition with its substrate, D-alanine. Binding of progesterone brought about the increase in both fluorescence intensity and fluorescence polarization of FAD, which indicates that the environment surrounding FAD chromophore is modified due to a conformational change in the apoenzyme. 2. Ethinyl estradiol, testosterone, testosterone propionate, corticosterone and aldosterone also inhibited the enzyme slightly in the same manner. Their binding also produced a slight increase in FAD fluorescence without decreasing the fluorescence polarization. 3. Cholesterol did not inhibit the enzyme, though it increased the fluorescence polarization of FAD. This indicates the binding of cholesterol with the enzyme at a site other than the substrate binding site.     

D-amino acid oxidase in mouse liver

1. An appreciable amount of D-amino acid oxidase was found in the extract of mouse liver by enzyme-linked immunosorbent assay (ELISA). 2. The content of the enzyme in the kidney and heart extracts was also measured by the assay.     

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

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