The standard amino acids except glycine can exist as either L- or D- optical isomers. An example of L- and D- forms of alanine is shown in Figure 1.8. The L-amino acids are utilized as building blocks for proteins synthesized by all living organisms. D-amino acids are important for bacteria as fundamental elements of the bacterial cell wall peptidoglycan layer, but their catabolism mechanism is not well under- stood [63]. Several D-amino acids are reported to be involved in disassembly of bacterial biofilms [64] and growth phase-dependent cell wall remodeling [65]. Moreover, specific D-amino acids have been dis- covered in many physiological processes in vertebrates, including humans [66]. Dunlop et al. identified D-aspartate in the brain and other tissues of mammals and suggested that it may play a role in regulating the development of these organs [67]. D-serine was identified at significant levels in rat brain, at about a third of the level of L-serine [68]. Further, D-serine in the rat brain is distributed in close association with N-methyl-D-aspartate (NMDA) and it may act as an endogenous agonist of the NMDA receptor in mam- malian brains [68].
The catabolism of D-amino acids has not been intensively studied when compared to those of L- amino acids. D-amino acids could be metabolized either directly or after conversion into the L- enantiomers. Conversion of L- and D-amino acids in living organisms is commonly catalyzed by racemases. Various amino-acid racemases have been identified in bacteria, archaea, and eucaryotes in- cluding mammals. These racemases are classified into two groups: pyridoxal 5’-phosphate (PLP) - dependent and -independent enzymes [66]. D-serine racemases and D-aspartate racemases are intensively studied in mammals due to their involvement in cell aging and neural signaling [69-70]. In bacteria, D- amino acids are deaminated by flavin-dependent oxidases or dehydrogenase, allowing the bacteria to grow using D-amino acids as the nutrient and energy source [71-73].
Many flavoenzymes have been identified in the catabolism pathway of D-amino acids. D-amino acid oxidase has been the most extensively studied member since its first discovery in 1930s [48]. This enzyme catalyzes the dehydrogenation of D-amino acids into their corresponding imino acids. The imino acids are converted to keto acids and ammonium through a non-enzymatical reaction, while the reduced enzyme cofactor FAD is oxidized by molecule oxygen. The structure of DAAO is composed of two do- mains, a FAD-binding domain and a substrate-binding domain (or interface domain) (Figure 1.9a) [74]. The cofactor FAD adopts the elongated conformation with the flavin ring located at the interface between the two domains. The enzyme active site is identified near the re face of the cofactor as shown in the structures of DAAO with ligands [74-75].
DAAO is widely distributed in nature, from microorganisms to mammalian tissues, such as brain, kidney, and liver. The studies on DAAO used to be difficult due to the low content in tissues and low sta- bility of this enzyme. However, investigation in DAAO has increased dramatically since mid 1990s be- cause of the importance of its physiological roles and development of genetic engineering which allows producing the enzyme in large quantity [76-77]. DAAO is able to catalyze the deamination of many D- amino acids. Therefore, it is involved in many physiological processes. In microorganisms, DAAO can provide D-amino acids as a source of carbon, nitrogen, and energy [78]. In mammalian cells, DAAO plays important roles in many processes such as neutral signaling and hormone secretion by regulating level of D-amino acids. One of the most important functions of DAAO is its regulation of D-serine, which is known to be an endogenous agonist of NMDA-receptors in mammalian brains [68]. Increased DAAO activity will lead to decreased level of D-serine, hence lowering the activity of NMDA-receptors. It is suggested that this regulation is related to the development of schizophrenia [76]. Moreover, DAAO is involved in the process of hormone secretion due to its regulation of D-Aspartate [77]. DAAO also regu- lates the levels of D-proline and D-alanine. The level of D-oxyproline has been suggested to be related to aging [79]. D-alanine is found to be important for intracellular osmotic pressure and increased level of D- alanine has been reported in the gray matter of Alzheimer’s patient [80-81].
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One of the major characteristics of DAAO is its high specificity to D-amino acids, while it is al- most inactive to the corresponding L-amino acids. The deamination of L-amino acids is catalyzed by an- other flavin-dependent oxidoreductase, L-Amino acid oxidase (LAAO), which accepts most of the 20 L- amino acids as substrates but does not react with D-amino acids [47]. The structures of LAAO from Rhodococcus opacus (roLAAO) with different ligands have been determined (Figure 1.9b) [47]. The
overall topology of roLAAO is very similar to that of DAAO from pig kidney, although they share very low sequence identity (15.3%). The two enzymes are both composed of a substrate-binding domain and a FAD-binding domain except that LAAO contains an extra helical domain (colored yellow in Figure 1.9b), which is involved in the dimerisation of the enzyme [47]. How these enzymes precisely recognize the stereospecificity in their substrates has become an interesting topic. Detailed comparison of the structures of DAAO and LAAO has revealed a mirror-symmetrical relationship through the plane perpendicular to the isoalloxazine ring between their active sites, which will be responsible for their different enantiomeric selectivity [45]. Meanwhile, similar composition and arrangement of the active site residues have been observed between the D- and L- amino acid oxidases, which may explain the similarities in their func- tions. Moreover, a different mode of mirror-symmetrical relationship for opposite stereospecificity has been observed between DAAO and flavocytochrome b2, which oxidizes L-lactate to pyruvate [82]. The active sites of the two enzymes are mirrored through the plane of isoalloxazine ring. The ligand binds to the re side of FAD in pDAAO, while it binds to the si side of FAD in flavocytochrome b2 [74].