Free d glutamate also has been found in

Free d-glutamate also has been found in various tissues of invertebrates and vertebrates including mammals (Han et al., 2011; Ariyoshi et al., 2017), fish (Kera et al., 2001) amphibians (Kera et al., 2001), birds (Kera et al., 2001), mollusks (Tarui et al., 2003; Patel et al., 2017) and arthropods (Corrigan, 1969; Sekimizu et al., 2005; Yoshikawa et al., 2011). It is currently known that d-glutamate plays a role in the mammalian central nervous systems (Kera et al., 1995; Mangas et al., 2007; Pan et al., 1993) and in muscle contractions of silkworm (Sekimizu et al., 2005), but many facets of its physiological effect remain unclear. At least two enzymes capable of degrading d-glutamate have been identified in animals (Ariyoshi et al., 2017; Katane et al., 2007, Katane et al., 2010; Meister et al., 1963; Rocca and Ghiretti, 1958; Sarower et al., 2004; Zaar et al., 2002). The catabolic enzyme d-aspartate oxidase that catalyzes the oxidative deamination of d-aspartate and d-glutamate with O2 to generate the corresponding 2-oxo acids, is found in many species of vertebrates and invertebrates (Katane et al., 2007, Katane et al., 2010; Rocca and Ghiretti, 1958; Sarower et al., 2004; Zaar et al., 2002). Another d-glutamate degrading enzyme, d-glutamate cyclase, that catalyzes conversion of d-glutamate to 5-oxo-d-proline, was initially purified from mammalian kidney and liver (Meister et al., 1963). Very recently, Ariyoshi et al. identified the d-glutamate cyclase gene from mouse and showed an increase in heart d-glutamate levels in the d-glutamate cyclase knockout mouse compared with wild-type mouse (Ariyoshi et al., 2017). In contrast, the d-glutamate biosynthetic pathway in animals is still unknown. Although it is considered that d-glutamate in animals is synthesized by an amino 4sc racemase, in parallel to the synthesis of d-aspartate and d-serine, the existence of glutamate racemase has never been reported in animals. Recently, we found 11 mammalian SerR homologous genes from eight invertebrate phyla using GenBank DNA databases (Uda et al., 2016). Recombinant proteins corresponding to the 11 mammalian SerR homologs showed serine and/or aspartate racemase activities and were identified by their maximum activity as SerR or AspR (Uda et al., 2016; Katane et al., 2016). Among these enzymes, some glutamate racemase (GluR) activity was detected in the black tiger prawn (Penaeus monodon) and the Pacific oyster (Crassostrea gigas) AspR enzymes (Uda et al., 2016). However, it is likely that these enzymes do not act as glutamate racemase enzymes in vivo, since their GluR activities are very weak. The hemichordate acorn worm (Saccoglossus kowalevskii) is a marine invertebrate that is an important model organism to understand developmental processes and evolution of the chordate body plan, and its genome and transcriptome sequence are now available (Simakov et al., 2015; Freeman Jr et al., 2008). Genome and transcriptome data revealed that S. kowalevskii has two mammalian SerR homologs, but the function of these genes was unknown. In the present study, we cloned, expressed and characterized these two SerR homologs. One of them showed significant racemase activity only for aspartate, which was in agreement with the characteristic features of the typical animal AspR. The other homolog showed significant and comparable activity against aspartate and glutamate and it was considered to be aspartate/glutamate racemase (Asp/GluR). This is the first report showing the presence of an enzyme found in animals with activity significant enough to synthesize d-glutamate in vivo.
Materials and methods
Results and discussion
Conclusion This study has determined the kinetic parameters of two recombinant SerR homologs from S. kowalevskii. The two enzymes have been identified as AspR and Asp/GluR on the basis of their catalytic efficiency. The S. kowalevskii Asp/GluR is the first reported enzyme that can synthesize d-glutamate in animals. The site-directed mutagenesis analysis has revealed that Ala156 located in the triple serine loop region, which is known to regulate the AspR activity, is required for glutamate recognition. One of the more significant findings from the present study is that d-glutamate is likely synthesized in vivo due to the kinetic parameters of S. kowalevskii Asp/GluR from its l-isomer similarly to d-serine and d-aspartate in animals. The second finding is that Asp/GluR has apparently evolved from SerR via AspR. Identifying the d-glutamate synthase enzyme in the present study will contribute to the future studies of d-glutamate metabolism in various animals. Moreover, the fact that SerR acquired substrate specificity towards aspartate or glutamate and evolved to AspR or GluR, raises the possibility that synthesis of other d-amino acids may be carried out by enzymes evolved from SerR. Unfortunately, our study did not include information on the presence of d-glutamate in S. kowalevskii because live specimens could not be obtained. Therefore, it is unknown if Asp/GluR synthesizes d-glutamate in vivo, and further studies need to be carried out in order to answer this question. Our findings suggest that the presence of Ala156 may be used to help identify GluR enzymes from genomic and transcriptomic data of various animals.