Endogenous D-amino acids are normally converted to L-amino acids by racemases [66]. In 2009, Li et. al reported a novel 2-component amino acid racemase involved in D-to-L inversion in Pseudomo- nas aeruginosa, which is an opportunistic human pathogen [83]. A dauBAR operon was found to be high-
ly induced by D-arginine from the DNA microarray analysis. Mutagenesis study showed that mutations at dauA or dauB prevented the bacteria from using D-arginine as sole carbon source. Further studies have
lyzes the conversion of D-arginine into 2-ketoarginine and ammonia, and L-arginine dehydrogenase, which is encoded by dauA and converts 2-ketoarginine to L-arginine (Figure 1.10) [83]. Enzymatic stud- ies on 19 D-amino acids have revealed that DADH has broad substrate specificity, with D-arginine and D-lysine as the two best substrates [45, 84].
In order to understand the reaction mechanism and substrate specificity of DADH, the crystal structure of DADH was determined at the atomic resolution of 1.06 Å, while the structures of DADH crystallized in the presence of iminoarginine, iminohistidine, iminolysine, iminomethionine, iminophenylalanine and iminoproline were also determined at the resolutions of 1.03 Å to 1.30 Å. Well- defined electron densities for the non-covalently bound FAD and imino intermediates of the reaction al- lowed detailed analysis of the enzyme active site. Alternative conformations have been observed for a loop region near the DADH active site, suggesting its involvement in substrate binding and product re- lease. Iminoarginine and iminohistidine bind to the active site in distinct modes, which is in agreement with detailed kinetic analysis on substrate specificity reported previously [84]. The structural characteris- tics described here will provide insights into substrate recognition and the catalytic reaction mechanism of DADH.
DADH contains one molecule of FAD as a cofactor and is composed of a substrate-binding do- main and a FAD-binding domain, while the active site is located at the interface of the two domains (Fig- ure 1.11a). Sequence of this enzyme showed that it shares very low sequence identity (<20%) with other FAD-containing enzymes such as L-proline dehydrogenase (18.5% [50]), DAAO (17.2%, [74]), and LAAO (16.4%, [47]). However, the three-dimensional structure of DADH highly resembles those of oth- er flavoenzymes (see detailed comparison in chapter 3). The FAD-binding domain of DADH adopts the rossmann fold that is predominant in the GR flavoenzyme family as discussed in above. It consists of a central six-stranded β-sheet surrounded by five α-helixes on one side and a three-stranded anti-parallel β-
sheet with two α-helixes on the other side (Figure 1.11b). The substrate binding domain is composed of
an eight-stranded β-sheet and two short antiparallel β-strands forming a sandwich surrounded by four α-
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Examination of the electron density map demonstrated that the reaction product imino acids were captured in the active sites of the determined crystal structures. Further comparison of structures of the ligand-free DADH and its complexes with imino acids has revealed that the protein active site undergoes major conformational change upon binding of the ligand. It possesses a ligand-free conformation in the free enzyme but shows a product-bound conformation upon binding of the ligand. Residues 50-56 were designated as an active site lid controlling the substrate accessibility to the active site. This active site lid structure has been reported in many flavoenzymes, such as D-amino acid oxidase (residues 216-228 [48]), pyranose 2-oxidase (residues 454-457 [85]), and choline oxidase (residues 64-95 [86]) (Figure 1.12). The loop composed of residues 216-228 in DAAO have been proposed to act as a lid that is able to switch from closed and open conformation to control substrate binding and product release [74]. This hypothesis is supported by the experiment that proteolysis of the peptide bond between loop residues 221-222 is pre- vented by inhibitor binding, suggesting a conformational alternation upon binding of a ligand [87]. The closed conformation of DAAO active site lid has been observed in several structures of DAAO in com- plexes of different ligands [74-75]. However, the open conformation has never been captured in crystal structure based on our knowledge. The ligand-free and product-bound conformations observed in the DADH helped confirm the hypothesis about this active site lid. Not only it is involved in ligand binding and releasing, it may also play a role in the substrate specificity of DADH [45].
DADH is characterized by broad substrate specificity, being able to oxidize basic and hydropho- bic D-amino acids of various sizes, but not reacting with acidic D-amino acids [45, 84]. D-amino acids are converted enzymatically to imino acids, which then dissociate from the enzyme and are hydrolyzed to keto acids in a nonenzymatic reaction. Steady-state kinetic studies with D-arginine or D-histidine as sub- strate have established a ping-pong bi-bi kinetic mechanism for this enzyme [88]. A product release ex- periment with D-arginine indicated that the release of imino product is partially rate limiting for the over- all turnover of the enzyme, which is in agreement with the observation that imino acids were trapped in the crystal structure. DADH has been suggested to be a true dehydrogenase due to its lack of oxygen reac- tivity [88]. Structural analysis suggested that the access of O2 to the flavin C4a atom is blocked by the
Ala46 and the absence of positive charge proximal to the flavin C4a and N1-C2 atoms is also responsible for its poor oxygen reactivity. A mutation of DAAO Gly52, which is located at the corresponding posi- tion of DADH Ala46, to Val leads to the loss of DAAO reactivity with oxygen due to the steric hindrance blocking the oxygen access [89]. Furthermore, substitution of the structurally equivalent Ala113 with Gly in L-galactono-γ-lactone dehydrogenase increased the reactivity of the reduced flavin with oxygen by about 400-fold [44].
The structural studies on DADH presented here may facilitate knowledge of the mechanism of catabolism of D-amino acids in vivo. The conformational change at the active site lid has been clearly shown in the atomic resolution structure of DADH, suggesting a role in controlling the substrate binding and product releasing. This active site lid has been proposed in other enzymes such as DAAO but with no direct evidence. The ionic interaction of iminoarginine with Glu87 in DADH is suggested to be critical for the enzyme specificity for basic D-amino acids. This is supported by the kinetic study showing that D-
arginine and D-lysine are the two best substrates and the kcat/KArg value is about 1000-fold higher than the
kcat/KHis value. Moreover, the structural analysis of DADH in complexes with hydrophobic imino acids revealed the hydrophobic residues responsible for the binding of bulky hydrophobic substrates. Further comparison of DADH with other functional related enzymes like DAAO and L-amino acids oxidase has revealed some key components and structural arrangement that are responsible for their specificity toward different enantiomers and different amino acids [45]. Detailed structural comparison of DADH with D- amino acid oxidase and D-aspartate oxidase indicated that although they share active site similarities in terms of recognition of D-amino acids, different arrangement and composition of the key structural fea- tures determine very distinct substrate specificities. Overall, the high-resolution structures of DADH will provide useful information for future studies of similar flavin-dependent enzymes.
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