Substitution of Arginine 120 in Human D-Amino Acid Oxidase Favors FAD-Binding and Nuclear Mistargeting.

Giulia Murtas,Silvia Sacchi,Loredano Pollegioni

doi:10.3389/fmolb.2019.00125

Abstract

The peroxisomal enzyme human D-amino acid oxidase (hDAAO) is attracting attention owing to its role in degrading D-serine, the main co-agonist of N-methyl D-aspartate receptors in brain, and its involvement in brain functions and diseases. Here, we focused on arginine 120, a residue located at the protein interface, 20 Å from the assumed second ligand-binding site, showing a different orientation of the side chain in the hDAAO-benzoate complex, and corresponding to Ser119 in rat DAAO, which is part of a putative nuclear translocation signal (NTS). By substituting Arg120 in hDAAO with a glutamate (to mimic the active NTS) or a leucine (to eliminate the positive charge) the protein conformation, thermal stability, and kinetic properties are slightly altered, while the dimeric structure and the ligand-binding properties are unchanged. The most relevant alteration in Arg120 variants is the strongest interaction with FAD. Nevertheless, the activity assayed at low D-serine and FAD concentrations (resembling physiological conditions) was quite similar for wild-type and Arg120 hDAAO variants. These results resemble the ones obtained substituting another residue located at the interface region (i.e., the W209R variant), indicating that substitutions at the monomer-monomer interface mainly affects the FAD binding in hDAAO. Indeed, U87 glioblastoma cells transiently transfected for hDAAO variants show that substitution of Arg120 favors mistargeting: the increase in cytosolic localization observed for the variants promotes nuclear targeting, especially for the R120E hDAAO, without affecting cell viability. Notably, mistargeting to the nucleus is an innate process as it is apparent for the wild-type hDAAO, too: whether such a process is related to specific pathologic processes is still unknown.

Highlights

Human D-amino acid oxidase is a FAD-dependent enzyme, which catalyzes the oxidative deamination of neutral D-amino acids into the corresponding α-keto acids, ammonia, and hydrogen peroxide (Pollegioni et al, 2007; Sacchi et al, 2012; Murtas et al, 2017)
U87 glioblastoma cells transiently overexpressing human D-amino acid oxidase (hDAAO) showed that the flavoenzyme is cytosolic before being delivered to peroxisomes: a time course of protein import was reported in Sacchi et al (2008, 2011)
The tertiary structure is largely preserved: only for the R120E hDAAO does alteration in the near-UV Circular dichroism (CD) and fluorescence spectra suggest that the orientation of aromatic residues has changed and is coupled to a decrease in the α-helix content

Summary

Introduction

Human D-amino acid oxidase (hDAAO, EC 1.4.3.3) is a FAD-dependent enzyme, which catalyzes the oxidative deamination of neutral D-amino acids into the corresponding α-keto acids, ammonia, and hydrogen peroxide (Pollegioni et al, 2007; Sacchi et al, 2012; Murtas et al, 2017). The mistargeting of the enzyme could be due to the presence of an NTS (TPx sequence corresponding to 117T-P-S119) that, after phosphorylation of Ser119, might activate the translocation to the nucleus (Chuderland et al, 2008; Luks et al, 2017a). Deletion of the C-terminal peroxisomal targeting signal 1 (PTS1) in both hDAAO and rDAAO resulted in a diffused nuclear and cytosolic distribution of the flavoenzyme (Luks et al, 2017b): nuclear localization was higher for rDAAO probably because a serine is phosphorylated more frequently than an arginine or was due to the different molecular masses of the two homologous enzymes (40 vs 80 kDa for rDAAO and hDAAO, respectively). Substitution of the putative tripeptide for nuclear translocation with the EPE sequence fully abolished nuclear import, independently of the presence of the PTS1 signal (Luks et al, 2017b)

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Results

Conclusion