DAAO is a flavine adenine dinucleotide-containing flavoenzyme and specifically catalyzes oxidative deamination of ᴅ-amino acids. There is only a single copy of daao in mammals. Daao in human is located at the chromosome 12q24 locus, and consists of 11 exons and a 1595 nt cDNA containing 347 codes [20, 21]. The similarity of DAAO coding sequence (CDS) in mouse (Mus musculus), rat (Rattus norvegicus), golden hamster (Mesocricetus auratus), grey hamster (Oryctolagus cuniculus), rabbit (Oryctolagus cuniculus), wild boar (Sus scrofa), human (Homo sapiens) is more than 90% (Figure S6), with slight difference on CDS length and protein size (Table 3).
Table 3
Comparison of DAAOs in different mammals
| mDAAO | rDAAO | pkDAAO | hDAAO |
Species | Mus musculus | Rattus norvegicus | Sus scrofa | Homo sapiens |
CDS length (bp) | 1035 | 1038 | 1041 | 1041 |
amino acid residues | 345 | 346 | 347 | 347 |
M.W. (kDa) | 38.65 | 38.82 | 39.34 | 39.47 |
FAD binding Kd | n.d | 0.30 µM | 0.22 µM | 8.0 µM |
Active site residues | Y222,Y226,R281 | Y223,Y227,R282 | Y224,Y228,R283 | Y224,Y228,R283 |
n.d: not determined.
The mouse daao gene is located on chromosome 5 (Figure S7) and consists of 11 exons. Among amino acid sequences of mDAAO included in the NCBI database, in addition to the natural mutant G181R [22], there were also other 5 differences in site (V/A64, K/N157, a glycine insertion at G172, H/R296 and F/H297) (Figure S8).
Aromatic carboxylic acid benzoic acid and its sodium salt were the first DAAO inhibitors discovered by humans. Although their effects were low, they were still commonly used as tool drug to study the biological functions of DAAO. Aromatic carboxylic acids can be divided into monocyclic and bicyclic parent nuclear molecular structure, such as 5-methylpyrazole-3-formic acid (generic name AS057278) and pyrrole-2-formic acid [23]. There are many bicyclic aromatic carboxylic acids, such as 4H-furano-[3,2-b] pyrrolo-5-carboxylic acid (Compound SUN) and thienopyrrolio-5-carboxylic acid (Compound 8) [24, 25], which belongs to the first class of aromatic carboxylic acid DAAO inhibitors. Compound SUN is a potent and specific DAAO inhibitor with IC50 values of 9-141 nM, but has little effect on human D-aspartate oxidase (IC50 values of 5-195 µM), which is the closest homologue of DAAO [26, 27]. The second generation is the benzoisoxazole group represented by 6-chloro-3-hydroxybenzoisoxazole (generic name CBIO) [28]. In the study of extracellular enzyme activity, the IC50 of CBIO on pDAAO was 188 nM, which was close to the first-generation inhibitor [29]. 3-hydroxyisoxazole of CBIO resembles an amino acid structure with the function similar to a carboxylic acid group of first-generation inhibitors [30].
In our recent studies, DAAO is specifically expressed in the peroxisome of astrocytes in rodent animal [31]. In MCAO-induced ischemic stroke, DAAO expression and enzyme activity in the penumbra of cerebral cortex also increased in a time-dependent manner with the continuous activation of astrocytes [32]. These results declared that the increment of cerebral DAAO expression and enzymic activity were causally associated with the development of neuronal destruction following ischemic insults. Therefore DAAO could be a potential target for treatment of many neuropathic diseases, including formalin-induced tonic pain, neuropathic pain, chronic morphine-induced analgesia tolerance and hyperalgesia, schizophrenia and cerebral ischemia.
The results indicated that the difference of mDAAO mutants between C57 and Balb/c in amino acid levels (V/A64, R/H295) could partly impact on mDAAO activity and their affinity with some inhibitors in vitro. Based on amino acid properties, mutations at these two sites have relatively limited impact on DAAO activity. Valine and alanine in V64A are both hydrophobic and non-polar amino acids, with only slight difference on steric hindrance of side chain and hydrophobicity. Arginine and histidine in R196H are similar amino acids with positive polarity.
This study successfully constructed a prokaryotic expression system for heterologous expression of DAAO in vitro, and clarified the expression and purification of DAAO in this system. Secondly, the ratio of Kcat/Km indicated that catalytic activity of DAAO in Balb/c was relatively lower. The IC50 value of C57 was less than others, which indicated that the affinity of C57 DAAO with DAAO inhibitor CBIO was higher. In total, the differences in amino acid levels at these two sites (V64A, R295H) partly impacted the catalytic efficiency of DAAO mutants and their affinity with some inhibitors in vitro.