Peptide name : D-amino-acid oxidase

Source/Organism : Not found

Linear/Cyclic : Not found

Chirality : D

Peptide length: 368

C-terminal modification: Not found

N-terminal modification : Free

Non-natural peptide information: None

Assay type : Not specified

Assay time : Not found

Activity : Not found

Cell line : Not found

Cancer type : Not found

Other activity : Not found

Amino acid composition bar chart :

Molecular mass : 40076.0027 Dalton

Aliphatic index : 0.827

Instability index : 44.8701

Hydrophobicity (GRAVY) : -0.308

Isoelectric point : 8.4438

Charge (pH 7) : 3.2776

Aromaticity : 0.073

Molar extinction coefficient (cysteine, cystine): (60390, 60765)

Hydrophobic/hydrophilic ratio : 1.12716763

hydrophobic moment : -0.388

Missing amino acid : None

Most occurring amino acid : G

Most occurring amino acid frequency : 35

Least occurring amino acid : M

Least occurring amino acid frequency : 4

Secondary structure fraction (Helix, Turn, Sheet): (0.3, 0.3, 0.3)

SMILES Notation: CC[C@H](C)[C@H](NC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)NC(=O)CNC(=O)[C@H](CC(=O)O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H]1CCCN1C(=O)[C@@H]1CCCN1C(=O)[C@H](CS)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCSC)NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H](NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H]1CCCN1C(=O)CNC(=O)[C@H](CC(=O)O)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CO)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@@H](N)CCSC)C(C)C)C(C)C)C(C)C)C(C)C)[C@@H](C)CC)[C@@H](C)CC)C(C)C)[C@@H](C)CC)C(C)C)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)C(C)C)C(C)C)[C@@H](C)O)[C@@H](C)O)[C@@H](C)CC)[C@@H](C)O)[C@@H](C)CC)C(C)C)[C@@H](C)O)[C@@H](C)O)C(C)C)[C@@H](C)O)[C@@H](C)O)C(C)C)[C@@H](C)O)C(C)C)C(C)C)[C@@H](C)O)[C@@H](C)CC)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(=O)N[C@@H](CCCNC(=N)N)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@H](C(=O)N[C@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(=N)N)C(=O)N1CCC[C@H]1C(=O)NCC(=O)NCC(=O)N[C@@H](CCC(=O)O)C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)NCC(=O)NCC(=O)N[C@H](C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)NCC(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(=O)O)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(=O)O)C(=O)NCC(=O)N[C@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=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1 : Chang YC, et al. Differences between Cryptococcus neoformans and Cryptococcus gattii in the Molecular Mechanisms Governing Utilization of D-Amino Acids as the Sole Nitrogen Source. PLoS One. 2015; 10:e0131865. doi: 10.1371/journal.pone.0131865

2 : Pollegioni L, et al. Studies on the structural and functional aspects of Rhodotorula gracilis D-amino acid oxidase by limited trypsinolysis. Biochem J. 1995; 310 ( Pt 2):577-83. doi: 10.1042/bj3100577

3 : Sacchi S, et al. Engineering the substrate specificity of D-amino-acid oxidase. J Biol Chem. 2002; 277:27510-6. doi: 10.1074/jbc.M203946200

4 : Abad S, et al. High-level expression of Rhodotorula gracilis D-amino acid oxidase in Pichia pastoris. Biotechnol Lett. 2011; 33:557-63. doi: 10.1007/s10529-010-0456-9

5 : Rosini E, et al. Optimization of D-amino acid oxidase for low substrate concentrations--towards a cancer enzyme therapy. FEBS J. 2009; 276:4921-32. doi: 10.1111/j.1742-4658.2009.07191.x

6 : Rosini E, et al. An antibody-based enzymatic therapy for cancer treatment: The selective localization of D-amino acid oxidase to EDA fibronectin. Nanomedicine. 2021; 36:102424. doi: 10.1016/j.nano.2021.102424

7 : Pollegioni L, et al. Kinetic mechanism of D-amino acid oxidases from Rhodotorula gracilis and Trigonopsis variabilis. J Biol Chem. 1993; 268:13850-7.

8 : Sacchi S, et al. Modulating D-amino acid oxidase substrate specificity: production of an enzyme for analytical determination of all D-amino acids by directed evolution. Protein Eng Des Sel. 2004; 17:517-25. doi: 10.1093/protein/gzh064

9 : Molla G, et al. Regulation of D-amino acid oxidase expression in the yeast Rhodotorula gracilis. Yeast. 2003; 20:1061-9. doi: 10.1002/yea.1023

10 : Kuan I, et al. Properties of Rhodotorula gracilis D-amino acid oxidase immobilized on magnetic beads through his-tag. J Biosci Bioeng. 2008; 105:110-5. doi: 10.1263/jbb.105.110

11 : Boselli A, et al. Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity. Biochimie. 2007; 89:360-8. doi: 10.1016/j.biochi.2006.10.017

12 : Piubelli L, et al. Conversion of the dimeric D-amino acid oxidase from Rhodotorula gracilis to a monomeric form. A rational mutagenesis approach. FEBS Lett. 2002; 526:43-8. doi: 10.1016/s0014-5793(02)03111-3

13 : Pollegioni L, et al. Cloning, sequencing and expression in E. coli of a D-amino acid oxidase cDNA from Rhodotorula gracilis active on cephalosporin C. J Biotechnol. 1997; 58:115-23. doi: 10.1016/s0168-1656(97)00142-9

14 : Alonso J, et al. D-amino-acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217. Microbiology (Reading). 1998; 144 ( Pt 4):1095-1101. doi: 10.1099/00221287-144-4-1095

15 : Pollegioni L, et al. Yeast D-amino acid oxidase: structural basis of its catalytic properties. J Mol Biol. 2002; 324:535-46. doi: 10.1016/s0022-2836(02)01062-8

16 : Umhau S, et al. The x-ray structure of D-amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-dependent substrate dehydrogenation. Proc Natl Acad Sci U S A. 2000; 97:12463-8. doi: 10.1073/pnas.97.23.12463

17 : Pernot P, et al. Characterization of a yeast D-amino acid oxidase microbiosensor for D-serine detection in the central nervous system. Anal Chem. 2008; 80:1589-97. doi: 10.1021/ac702230w

18 : Stegman LD, et al. Induction of cytotoxic oxidative stress by D-alanine in brain tumor cells expressing Rhodotorula gracilis D-amino acid oxidase: a cancer gene therapy strategy. Hum Gene Ther. 1998; 9:185-93. doi: 10.1089/hum.1998.9.2-185

19 : Perotti ME, et al. Expression of D-amino acid oxidase in Rhodotorula gracilis under induction conditions: a biochemical and cytochemical study. Eur J Cell Biol. 1991; 55:104-13.

20 : Pollegioni L, et al. Catalytic properties of D-amino acid oxidase in cephalosporin C bioconversion: a comparison between proteins from different sources. Biotechnol Prog. 2004; 20:467-73. doi: 10.1021/bp034206q

Paper title : Differences between Cryptococcus neoformans and Cryptococcus gattii in the Molecular Mechanisms Governing Utilization of D-Amino Acids as the Sole Nitrogen Source.

Doi : https://doi.org/10.1371/journal.pone.0131865

Abstract : The ability to grow on media containing certain D-amino acids as a sole nitrogen source is widely utilized to differentiate Cryptococcus gattii from C. neoformans. We used the C. neoformans H99 and C. gattii R265 strains to dissect the mechanisms of D-amino acids utilization. We identified three putative D-amino acid oxidase (DAO) genes in both strains and showed that each DAO gene plays different roles in D-amino acid utilization in each strain. Deletion of DAO2 retarded growth of R265 on eleven D-amino acids suggesting its prominent role on D-amino acid assimilation in R265. All three R265 DAO genes contributed to growth on D-Asn and D-Asp. DAO3 was required for growth and detoxification of D-Glu by both R265 and H99. Although growth of H99 on most D-amino acids was poor, deletion of DAO1 or DAO3 further exacerbated it on four D-amino acids. Overexpression of DAO2 or DAO3 enabled H99 to grow robustly on several D-amino acids suggesting that expression levels of the native DAO genes in H99 were insufficient for growth on D-amino acids. Replacing the H99 DAO2 gene with a single copy of the R265 DAO2 gene also enabled its utilization of several D-amino acids. Results of gene and promoter swaps of the DAO2 genes suggested that enzymatic activity of Dao2 in H99 might be lower compared to the R265 strain. A reduction in virulence was only observed when all DAO genes were deleted in R265 but not in H99 indicating a pathobiologically exclusive role of the DAO genes in R265. These results suggest that C. neoformans and C. gattii divergently evolved in D-amino acid utilization influenced by their major ecological niches.

Paper title : Studies on the structural and functional aspects of Rhodotorula gracilis D-amino acid oxidase by limited trypsinolysis.

Doi : https://doi.org/10.1042/bj3100577

Abstract : The structure-function relationships of purified Rhodotorula gracilis D-amino acid oxidase (in its holo-, apo- and holo-enzyme-benzoate complex forms) was analysed by digestion with trypsin. In all cases trypsin cleaves this 80 kDa dimeric enzyme at the C-terminal region, since the peptide bonds sensitive to proteinase attack are clustered in this region. Digestion of native enzyme with trypsin produced a nicked and truncated form of 38.3 kDa containing two polypeptides of 34 and 5 kDa starting from Met1 and Ala319 respectively, and with detachment of the Thr306-Arg318 and Glu365-Leu368 peptides. Our results show that this 'core', folded into a compact structure, is catalytically competent. The acquisition of this nicked form was marked by a shift from a dimeric to a monomeric active enzyme, a result never previously obtained. The deleted sequences, Thr306-Arg318 and Glu365-Leu368, are essential for the monomer-monomer interaction, and, in particular, the region encompassing Thr306-Arg318 should play an essential role in the dimerization process. interestingly, the Ser308-Lys321 sequence present in the lost peptide corresponds to a sequence not present in other known D-amino acid oxidases [Faotto, Pollegioni, Ceciliani, Ronchi and Pilone (1995) Biotechnol. Lett. 17, 193-198]. A role of the cleaved-off region for the thermostabilization of the enzyme is also discussed.

Paper title : Engineering the substrate specificity of D-amino-acid oxidase.

Doi : https://doi.org/10.1074/jbc.M203946200

Abstract : The high resolution crystal structure of D-amino-acid oxidase (DAAO) from the yeast Rhodotorula gracilis provided us with the tool to engineer the substrate specificity of this flavo-oxidase. DAAO catalyzes the oxidative deamination of D-amino acids, with the exception of D-aspartate and D-glutamate (which are oxidized by D-aspartate oxidase, DASPO). Following sequence homology, molecular modeling, and simulated annealing docking analyses, the active site residue Met-213 was mutated to arginine. The mutant enzyme showed properties close to those of DASPO (e.g. the oxidation of D-aspartate and the binding of l-tartrate), and it was still active on D-alanine. The presence of an additional guanidinium group in the active site of the DAAO mutant allowed the binding (and thus the oxidation) of D-aspartate, but it was also responsible for a lower catalytic activity on D-alanine. Similar results were also obtained when two additional arginines were simultaneously introduced in the active site of DAAO (M213R/Y238R mutant, yielding an architecture of the active site more similar to that obtained for the DASPO model), but the double mutant showed very low stability in solution. The decrease in maximal activity observed with these DAAO mutants could be due to alterations in the precise orbital alignment required for efficient catalysis, although even the change in the redox properties (more evident in the DAAO-benzoate complex) could play a role. The rational design approach was successful in producing an enzymatic activity with a new, broader substrate specificity, and this approach could also be used to develop DAAO variants suitable for use in biotechnological applications.

Paper title : High-level expression of Rhodotorula gracilis D-amino acid oxidase in Pichia pastoris.

Doi : https://doi.org/10.1007/s10529-010-0456-9

Abstract : By combining gene design and heterologous over-expression of Rhodotorula gracilis D-amino acid oxidase (RgDAO) in Pichia pastoris, enzyme production was enhanced by one order of magnitude compared to literature benchmarks, giving 350 kUnits/l of fed-batch bioreactor culture with a productivity of 3.1 kUnits/l h. P. pastoris cells permeabilized by freeze-drying and incubation in 2-propanol (10% v/v) produce a highly active (1.6 kUnits/g dry matter) and stable oxidase preparation. Critical bottlenecks in the development of an RgDAO catalyst for industrial applications have been eliminated.

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

Doi : https://doi.org/10.1111/j.1742-4658.2009.07191.x

Abstract : 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.

Paper title : An antibody-based enzymatic therapy for cancer treatment: The selective localization of D-amino acid oxidase to EDA fibronectin.

Doi : https://doi.org/10.1016/j.nano.2021.102424

Abstract : In order to generate an antibody directed enzyme prodrug therapy, here we designed a chimeric protein by fusing the F8 antibody that recognizes the EDA of fibronectin (expressed on the tumor neovasculature) and an evolved variant of the ROS-generating enzyme D-amino acid oxidase (DAAO). The F8(scFv)-DAAO-Q144R recombinant protein is expressed by both CHO-S and E. coli cells. The F8(scFv)-DAAO-Q144R from E. coli cells is fully soluble, shows a high specific activity, is more thermostable in blood than the native DAAO, possesses a binding affinity for EDA well suited for efficient tumor accumulation, and localizes in tumor tissues. Notably, the F8(scFv)-DAAO-Q144R conjugate generates a stronger cytotoxicity to tumor cells than the native enzyme, especially when an inhibitor of heme oxygenase-1 (HO-1) is used, making it a promising candidate for a selective antitumor oxidative therapy controlled by the substrate addition, in the so called "activity on demand", thus sparing normal tissue from damage.

Paper title : Kinetic mechanism of D-amino acid oxidases from Rhodotorula gracilis and Trigonopsis variabilis.

Doi : https://doi.org/Not available

Abstract : The reaction of two D-amino acid oxidases from the yeasts Rhodotorula gracilis and Trigonopsis variabilis with the substrates alanine and valine in their 2-1H and 2-2H forms was studied employing the stopped-flow spectrophotometric technique. The turnover numbers at infinite substrate and oxygen concentrations were: 20,700/4,250 and 1,730/360 ([2-1H]/[2-2H]alanine and valine, respectively) for the Rhodotorula and 3,150/440 and 2,500/520 ([2-1H]/[2-2H]alanine and valine, respectively) for the Trigonopsis enzymes. The rates of anaerobic enzyme flavin reduction were 20,100/4,000 and 1,820/350 ([2-1H]/[2-2H]alanine and valine, respectively) for the Rhodotorula and 3,470/350 and 2,460/480 ([2-1H]/[2-2H]alanine and valine, respectively) for the Trigonopsis enzymes. The isotope effects on enzyme reduction were 5.0 and 5.2 for Rhodotorula and 9.9 and 5.1 for Trigonopsis D-amino acid oxidases with alanine and valine, respectively. This suggests that the intrinsic isotope effect on rupture of the substrate alpha-C-H bond can be as high as 10. The rate-determining step corresponds to the enzyme reductive half-reaction in contrast to the mammalian kidney enzyme where it is the product release from oxidized enzyme (Massey, V., and Gibson, Q.H. (1964) Fed. Proc. 23, 18-29). Upon anaerobic reaction with substrate, the yeast enzymes do not form the transient long wavelength absorbing species which are characteristic of the mammalian protein. This is due only in part to rapid dissociation of iminoacid product and is ascribed to intrinsic differences between the charge-transfer complexes of reduced enzyme flavin and product of the yeast as compared to the mammalian enzyme. With the Trigonopsis enzyme the flavin radical anion appears to be strongly stabilized and can be produced quantitatively.

Paper title : Modulating D-amino acid oxidase substrate specificity: production of an enzyme for analytical determination of all D-amino acids by directed evolution.

Doi : https://doi.org/10.1093/protein/gzh064

Abstract : Recent research on the flavoenzyme D-amino acid oxidase from Rhodotorula gracilis (RgDAAO) has revealed new, intriguing properties of this catalyst and offers novel biotechnological applications. Among them, the reaction of RgDAAO has been exploited in the analytical determination of the D-amino acid content in biological samples. However, because the enzyme does not oxidize acidic D-amino acids, it cannot be used to detect the total amount of D-amino acids. We now present the results obtained using a random mutagenesis approach to produce RgDAAO mutants with a broader substrate specificity. The libraries of RgDAAO mutants were generated by error-prone PCR, expressed in BL21(DE3)pLysS Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. Five random mutants that have a 'modified' substrate specificity, more useful for the analytical determination of the entire content of D-amino acids than wild-type RgDAAO, have been isolated. With the only exception of Y223 and G199, none of the effective amino acid substitutions lie in segments predicted to interact directly with the bound substrate. The substitutions appear to cluster on the protein surface: it would not have been possible to predict that these substitutions would enhance DAAO activity. We can only conclude that these substitutions synergistically generate small structural changes that affect the dynamics and/or stability of the protein in a way that enhances substrate binding or subsequently catalytic turnover.

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

Doi : https://doi.org/10.1002/yea.1023

Abstract : 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.

Paper title : Properties of Rhodotorula gracilis D-amino acid oxidase immobilized on magnetic beads through his-tag.

Doi : https://doi.org/10.1263/jbb.105.110

Abstract : D-amino acid oxidase catalyzes one of the key steps in the production of semisynthetic cephalosporins. We expressed and purified recombinant Rhodotorula gracilis D-amino acid oxidase with C-terminal his-tags. This engineered enzyme was immobilized onto Ni(2+)-chelated nitrilotriacetic acid magnetic beads through the interaction between his-tag and Ni(2+). The kinetic constants, storage properties, and the reusability of the immobilized d-amino acid oxidase were determined. The effects of temperature, pH, and hydrogen peroxide on the activity of immobilized d-amino acid oxidase were also studied. The highest activity recovery was 75%. Thermal stability was improved after immobilization; the relative activity of the immobilized enzyme was 56% whereas the free enzyme was completely inactivated after incubation at 50 degrees C for 1 h. In the presence of 10 mM hydrogen peroxide, the immobilized enzyme did not show a rapid loss of activity during the first 2 h of incubation, which was observed in the case of the free enzyme; the residual activity of the immobilized enzyme after 9 h was 72% compared with 22% of the free form. The long-term storage stability was improved; the residual activity of the immobilized enzyme was 74% compared with 20% of the free enzyme when stored at room temperature for 10 d. The immobilized form retained 37% of its initial activity after 20 consecutive reaction cycles.

Paper title : Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity.

Doi : https://doi.org/10.1016/j.biochi.2006.10.017

Abstract : D-amino acid oxidase (DAAO) is a flavoprotein that catalyzes stereospecifically the oxidative deamination of D-amino acids. The wild-type DAAO is mainly active on neutral D-amino acids, while basic D-amino acids are poor substrates and the acidic ones are virtually not oxidized. To present a comprehensive picture of how the active site residues can modulate the substrate specificity a number of mutants at position M213, Y223, Y238, R285, S335, and Q339 were prepared in the enzyme from the yeast Rhodotorula gracilis. All DAAO mutants have spectral properties similar to those of the wild-type enzyme and are catalytically active, thus excluding an essential role in catalysis; a lower activity on neutral and basic amino acids was observed. Interestingly, an increase in activity and (k(cat)/K(m))(app) ratio on D-aspartate was observed for all the mutants containing an additional charged residue in the active site. The active site of yeast DAAO appears to be a highly evolved scaffold built up through evolution to optimize the oxidative deamination of neutral D-amino acids without limiting its substrate specificity. It is noteworthy, that introduction of a sole, additional, positively charged residue in the active site is sufficient to optimize the reactivity on acidic D-amino acids, giving rise to kinetic properties similar to those of D-aspartate oxidase.

Paper title : Conversion of the dimeric D-amino acid oxidase from Rhodotorula gracilis to a monomeric form. A rational mutagenesis approach.

Doi : https://doi.org/10.1016/s0014-5793(02)03111-3

Abstract : The relevance of the dimeric state for the structure/function relationships of Rhodotorula gracilis D-amino acid oxidase (RgDAAO) holoenzyme has been investigated by rational mutagenesis. Deletion of 14 amino acids in a surface loop (connecting beta-strands 12 and 13) transforms RgDAAO from a dimeric protein into a stable monomer. The mutant enzyme is still catalytically competent and retains its binding with the FAD coenzyme. Dimerization has been used by this flavoenzyme in evolution to achieve maximal activity, a tighter interaction between the protein moiety and the coenzyme, and higher thermal stability.

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

Doi : https://doi.org/10.1016/s0168-1656(97)00142-9

Abstract : 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 (approximately 0.5 U mg-1 protein) with a fermentation yield, in terms of DAAO units, of 800 U l-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 nucleotide sequence databases under accession number.

Paper title : D-amino-acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217.

Doi : https://doi.org/10.1099/00221287-144-4-1095

Abstract : The complete nucleotide sequence of the DAO1 gene encoding D-amino-acid oxidase (DAAO) in the yeast Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217 has been determined. The primary structure of DAAO was deduced from the nucleotide sequence of a cDNA clone that covered the entire amino acid coding sequence. Comparison of cDNA and genomic sequences of DAO1 revealed the presence of five introns. Because this is the first gene of strain ATCC 26217 that has been cloned so far, the nucleotide sequences of these introns were compared to those from other fungi. Upstream of the structural gene there was a stretch of C + T-rich DNA similar to that found in the promoter region of a number of yeast genes. The cDNA gene, which encoded a protein of 368 amino acids (molecular mass 40 kDa), was overexpressed in Escherichia coli under the control of the strong lipoprotein promoter. Interestingly, a significant fraction (13-62%) of the total DAAO activity was recovered in its apoenzyme form, the percentage depending on the culture conditions. This fact allowed a rapid purification of the recombinant DAAO by affinity chromatography. The high level of expression achieved in E. coli and the possibility of modifying its catalytic properties by protein engineering provide a new model for the study of this enzyme.

Paper title : Yeast D-amino acid oxidase: structural basis of its catalytic properties.

Doi : https://doi.org/10.1016/s0022-2836(02)01062-8

Abstract : The 3D structure of the flavoprotein D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis (RgDAAO) in complex with the competitive inhibitor anthranilate was solved (resolution 1.9A) and structural features relevant for the overall conformation and for catalytic activity are described. The FAD is bound in an elongated conformation in the core of the enzyme. Two anthranilate molecules are found within the active site cavity; one is located in a funnel forming the entrance, and the second is in contact with the flavin. The anchoring of the ligand carboxylate with Arg285 and Tyr223 is found for all complexes studied. However, while the active site group Tyr238-OH interacts with the carboxylate in the case of the substrate D-alanine, of D-CF(3)-alanine, or of L-lactate, in the anthranilate complex the phenol group rotates around the C2-C3 bond thus opening the entrance of the active site, and interacts there with the second bound anthranilate. This movement serves in channeling substrate to the bottom of the active site, the locus of chemical catalysis. The absence in RgDAAO of the "lid" covering the active site, as found in mammalian DAAO, is interpreted as being at the origin of the differences in kinetic mechanism between the two enzymes. This lid has been proposed to regulate product dissociation in the latter, while the side-chain of Tyr238 might exert a similar role in RgDAAO. The more open active site architecture of RgDAAO is the origin of its much broader substrate specificity. The RgDAAO enzyme forms a homodimer with C2 symmetry that is different from that reported for mammalian D-amino acid oxidase. This different mode of aggregation probably causes the differences in stability and tightness of FAD cofactor binding between the DAAOs from different sources.

Paper title : The x-ray structure of D-amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-dependent substrate dehydrogenation.

Doi : https://doi.org/10.1073/pnas.97.23.12463

Abstract : Flavin is one of the most versatile redox cofactors in nature and is used by many enzymes to perform a multitude of chemical reactions. d-Amino acid oxidase (DAAO), a member of the flavoprotein oxidase family, is regarded as a key enzyme for the understanding of the mechanism underlying flavin catalysis. The very high-resolution structures of yeast DAAO complexed with d-alanine, d-trifluoroalanine, and l-lactate (1.20, 1.47, and 1.72 A) provide strong evidence for hydride transfer as the mechanism of dehydrogenation. This is inconsistent with the alternative carbanion mechanism originally favored for this type of enzymatic reaction. The step of hydride transfer can proceed without involvement of amino acid functional groups. These structures, together with results from site-directed mutagenesis, point to orbital orientation/steering as the major factor in catalysis. A diatomic species, proposed to be a peroxide, is found at the active center and on the Re-side of the flavin. These results are of general relevance for the mechanisms of flavoproteins and lead to the proposal of a common dehydrogenation mechanism for oxidases and dehydrogenases.

Paper title : Characterization of a yeast D-amino acid oxidase microbiosensor for D-serine detection in the central nervous system.

Doi : https://doi.org/10.1021/ac702230w

Abstract : d-Serine is an endogenous ligand for N-methyl-d-aspartate (NMDA) receptors, and alterations in its concentration have been related to several brain disorders, especially schizophrenia. It is therefore an important target neuromodulator for the pharmaceutical industry. To monitor d-serine levels in vivo, we have developed a microbiosensor based on cylindrical platinum microelectrodes, covered with a membrane of poly-m-phenylenediamine (PPD) and a layer of immobilized d-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO). By detecting the hydrogen peroxide produced by enzymatic degradation of d-serine, this microbiosensor shows a detection limit of 16 nM and a mean response time of 2 s. Interferences by ascorbic acid, uric acid, l-cysteine, and by biogenic amines and their metabolites are rejected at more than 97% by the PPD layer. Although several d-amino acids are potential substrates for RgDAAO, d-serine was the only endogenous substrate present in sufficient concentration to be detected by our microbiosensor in the central nervous system. When implanted in the cortex of anesthetized rats, this microbiosensor detected the increase in concentration of d-serine resulting from its diffusion across the blood-brain barrier after an intraperitoneal injection. This new device will make it possible to investigate in vivo the variations in d-serine concentrations occurring under normal and pathological conditions and to assess the pharmacological potency of new drugs designed to impact d-serine metabolism.

Paper title : Induction of cytotoxic oxidative stress by D-alanine in brain tumor cells expressing Rhodotorula gracilis D-amino acid oxidase: a cancer gene therapy strategy.

Doi : https://doi.org/10.1089/hum.1998.9.2-185

Abstract : Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) generated in the stereoselective deamination of D-amino acids catalyzed by D-amino acid oxidase (DAAO). H2O2 readily crosses cellular membranes and damages DNA, proteins, and lipids. The scarcity of DAAO substrates in mammalian organisms and its co-localization with catalase in the peroxisomal matrix suggested that the cytotoxicity of ROS could be harnessed by administration of D-amino acids to tumor cells ectopically expressing DAAO in the cytoplasm. To evaluate this hypothesis, the cDNA encoding the highly active DAAO from the red yeast Rhodotorula gracilis was mutated to remove the carboxy-terminal peroxisomal targeting sequence. A clonal line of 9L glioma cells stably transfected with this construct (9Ldaao17) was found to synthesize active R. gracilis DAAO. Exposure of 9Ldaao17 cells to D-alanine resulted in cytotoxicity at concentrations that were nontoxic to parental 9L cells. Depletion of cellular glutathione further sensitized 9Ldaao17 cells to D-alanine (D-Ala). This result, combined with stimulation of pentose phosphate pathway activity and the production of extracellular H2O2 by 9Ldaao17 cells incubated with D-alanine implicates oxidative stress as the mediator of cytotoxicity. These results demonstrate that expression of R. gracilis DAAO in tumor cells confers chemosensitivity to D-alanine that could be exploited as a novel cancer gene therapy paradigm.

Paper title : Expression of D-amino acid oxidase in Rhodotorula gracilis under induction conditions: a biochemical and cytochemical study.

Doi : https://doi.org/Not available

Abstract : D-amino acid oxidase is expressed to a high level in the yeast Rhodotorula gracilis (0.3% of total cell protein) through induction by D-alanine in a defined growth medium. Monospecific polyclonal antibodies against pure enzyme were obtained. Western blot analysis showed that the enzyme is synthesized as the mature polypeptide. The localization of the enzyme was investigated by immunoelectron microscopy using the postembedding immunogold technique and by submicroscopic enzyme cytochemistry. D-Amino acid oxidase was detected in peroxisomes, and quantitation of immunoelectron microscopic data indicated that the enzyme is exclusively confined to these organelles. Immunoelectron microscopic observations are in complete agreement with biochemical data showing that the enzyme is not expressed in the absence of D-alanine. Morphometric analysis demonstrated that induction of D-amino acid oxidase synthesis is associated with a 241% increase of peroxisome volume density and with a 31% increase of peroxisome size as compared to cells grown on non-inducing medium.

Paper title : Catalytic properties of D-amino acid oxidase in cephalosporin C bioconversion: a comparison between proteins from different sources.

Doi : https://doi.org/10.1021/bp034206q

Abstract : Lacking an efficient process to produce 7-aminocephalosporanic acid from cephalosporin C in a single step, d-amino acid oxidase (DAAO) is of foremost importance in the industrial, two-step process used for this purpose. We report a detailed study on the catalytic properties of the three available DAAOs, namely, a mammalian DAAO and two others from yeast (Rhodotorula gracilis and Trigonopsis variabilis). In comparing the kinetic parameters determined for the three DAAOs, with both cephalosporin C and d-alanine as substrate, the catalytic efficiency of the two enzymes from microorganism is at least 2 orders of magnitude higher than that of pig kidney DAAO. Furthermore, the mammalian enzyme is more sensitive to product inhibition (from hydrogen peroxide and glutaryl-7-aminocephalosporanic acid). Therefore, enzymes from microorganisms appear to be by far more suitable catalysts for bioconversion, although some different minor differences are present between them (e.g., a higher activity of the R. gracilis enzyme when the bioconversion is carried out at saturating oxygen concentration). The mammalian DAAO, even being a poor catalyst, is more stable with respect to temperature than the R. gracilis enzyme in the free form. In any case, for industrial purposes DAAO is used only in the immobilized form where a strong enzyme stabilization occurs.