The crystal structure of D-amino acid oxidase with a substrate analog, o-aminobenzoate

Abstract

Since the discovery of D-amino acid oxidase (DAO) in 1935, many studies have been conducted without clarifying its 3D structure for a long time. In 1996, the crystal structure of DAO was determined, and it was shown that the catalytic bases required for the two catalytic mechanisms were not present in the active site. The crystal structure of DAO in complex with o-aminobenzoate was solved and is used for modeling Michaelis complex. The Michaelis complex model provided structural information leading to a new mechanism for reductive half-reaction of DAO. Currently, DAO is being researched for medical and applied purposes.

Abbreviations

 
• DAO

  D-amino acid oxidase

 
• FAD

  flavin adenine dinuculeotide

 
• HOMO

  highest occupied molecular orbital

 
• LUMO

  lowest unoccupied molecular orbital

D-Amino acid oxidase [D-amino acid:O₂ oxidoreductase (deaminating), EC 1.4.3.3] (DAO) was discovered by Hans Krebs in 1935 as the first mammalian flavoenzyme and the first flavin adenine dinuculeotide (FAD) enzyme (1). DAO is widely distributed in living organisms, ranging from microbes to mammals. The physiological significance of DAO remained cryptic because D-amino acids had long been considered foreign to organisms, particularly to higher animals. But currently, it has been revealed that D-amino acids occur in various organisms including mammals and play critical roles in regulating neural activities in vivo (2). The porcine kidney DAO is the most extensively studied among those found in various species and is the prototype of flavin-dependent oxidases. DAO catalyses the dehydrogenation of a D-amino acid, producing an imino acid that is subsequently hydrolyzed nonenzymatically to an α-ketoacid and ammonia. The reduced enzyme is reoxidized by molecular oxygen, generating hydrogen peroxide (Scheme 1). Before the active structure of DAO was clarified, reductive half-reaction was explained mainly as two alternative mechanisms. One is ‘carbanion mechanism’ (3); the other is ‘concerted mechanism’ (4). The former characterizes itself in the formation of a substrate abstraction by a protein catalytic base. The latter describes the reductive half-reaction as a concerted process of α-proton abstraction by a protein catalytic base and an electron transfer from the amino nitrogen to flavin. Either of the two mechanisms presupposes an amino acid residue, acting as a base to abstract substrate α-proton. In 1996, two groups published about the structure of DAO independent of each other (5,  6). It was clarified that there are no amino acid residues that are candidates for the catalytic base around the active site of DAO. o-Aminobenzoate (OAB) carry both amino and carboxyl group within a molecule, as does the substrate D-amino acid. Futhermore, when OAB is bound to DAO, spectral changes were observed due to the charge–transfer interaction between OAB and flavin, and the charge–transfer nature of the interaction in the DAO-OAB complex was proven using Raman spectroscopy with excitation of the absorption band (7). Therefore, to obtain a new Michaelis complex model that proposes a new catalytic mechanism, the crystal structure of the DAO-OAB complex was determined (8).

Yellow crystals of DAO were obtained with benzoate bound to the active site. Therefore, crystals of the DAO-OAB complex were obtained by the soaking methods. X-ray diffraction data were collected using an x-ray beam of wavelength 1.0 Å at 283 K on Fuji Imaging Plates with a screenless Weissenberg camera (9) at the BL6A, PF and KEK. The refinement of the DAO-OAB complex structure was initiated using coordinates DAO-benzoate complex determined at 2.5 Å resolution (unpublished result) using the program XPLOR (10), and manual adjustment and rebuilding of the model was performed using the program O (11).

The overall dimeric structure of the DAO-OAB complex with an elongated ellipsoidal shape was shown in Fig. 1. The two subunits, which are related through a non-crystallographic twofold axis, do no intrude into each other extensively. The active site is found in the area between the two domains; both flavin ring and OAB are found within this pocket and the wall of the distorted barrel. Figure 2 shows the geometrical arrangement of flavin, OAB and the residues constituting the active site and those in its neighbourhood, together with the hydrogen bonding network associated with flavin and OAB. The OAB lies on the ‘re’-face of the flavin ring system in accordance with the ‘re’-face specificity of DAO (14). The ‘si’-face is covered by the hydrophobic stretch blocking access of a substrate on the ‘si’-face. The carboxylate of OAB makes a salt bridge with the guanidino group of Arg283 and a hydrogen bond with the hydroxyl of Ty228. The amino group of OAB is hydrogen bonded with the backbone carbonyl of Gly313, which also forms a hydrogen bond with a bound water molecule that is further stabilized by two hydrogen bonds with Gln53 backbone carbonyl and with Tyr224 hydroxyl. The alignment between OAB and flavin is in such a way that C(2)-N of OAB overlaps with O=C (4) of flavin and the carboxyl carbon of OAB overlaps with flavin N (5). The charge–transfer interaction is generally attained by the overlap between the highest occupied molecular orbital (HOMO) of an electron donor and the lowest unoccupied molecular orbital (LUMO) of an electron acceptor. Therefore, there is an interaction between the HOMO of OAB and the LUMO of flavin (13). OAB has amino and carboxyl groups just like a substrate D-amino acid and is not oxidized by DAO but forms a charge–transfer complex with flavin. Thus, it can be interpreted as being partially oxidized form of charge transfer. Therefore, DAO-D-leucine Michaelis complex model prepared by replacing the substrate D-leucine with OAB-binding pocket was made (Fig. 3). Molecular mechanics simulation was done by energy minimization method using program XPLOR (10). The carboxylate group of D-leucine forms a salt bridge with the cationic guanidino group of Arg283 and a hydrogen bond with Tyr228 hydroxyl. The amino group does not have a salt-bridge but exits as a neutral form. As no negative charge is found within the active site, and the neutral amino group fits well within the hydrophobic environment. The amino group is hydrogen-bonded with O of Gly313, the lone pair orbital of the amino nitrogen is oriented towards C(4a) of flavin and the α-hydrogen can approach the lone pair of flavin N (5).

A new mechanism was proposed based on this DAO-D-leucine Michaelis complex model and the accumulation of enzymatic experimental results. In the DAO-catalysed reductive half-reaction, the C α-H bond is cleaved and α-hydrogen is removed as a result. Here, it is important to decide whether the α-hydrogen is released as a proton, a hydrogen atom or a hydride. When β-chloro-D-alanine is allowed to react with DAO, it undergoes elimination of hydrogen chloride in addition to normal oxidation yielding pyruvate and chloropyruvate, respectively. In the elimination reaction, substrate α-hydrogen behaves as a proton (15,  16). It is concluded that the substrate α-hydrogen behaves as a proton in the normal oxidation, i.e. reductive half-reaction of DAO, in the elimination reaction. This behaviour of α-hydrogen as a proton together with Michaelis complex model can be integrated into the reaction processes of electron transfer from the lone pair of the substrate amino nitrogen to flavin N (5), which, instead of an amino acid residue, acts as a proton-abstracting catalytic base. Thus, two possible mechanisms, the ‘electron-proton-electron mechanisms’ and the ‘ionic mechanism’ for the reductive half-reaction of DAO, were proposed.

DAO regulating the levels of neuromodulation D-serine, and DAO inhibitors are expected to be a potential treatment for schizophrenia. Additionally, the ability to redesign the specificity, oxygen affinity and stability of DAO substrates through protein engineering caused the growing use of DAO as a biosensor and biocatalyst. Many papers on DAO are still being published annually. About 40 DAO structures are currently registered in PDB, including complexes with various substrates and inhibitors, and high-resolution structures, with more to come.

DAO was the first enzyme to be phase-determined by isomorphous replacement method by heavy atom derivatization in my laboratory, and I recall that there was excitement when the electron density map of the phosphate group of FAD was confirmed because it was ensured that the phase had been determined. I also remember that Prof. Retsu Miura was pleased when the structure was determined, since DAO research was his life’s work. Prof. Miura contributed many papers to the Journal of Biochemistry, based on the idea that the value of the journal should be improved by Japanese researchers submitting many good papers. It is also because of this background that the 1AN9 structure was chosen this time.

References