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glutamate racemase from the acorn worm Saccoglossus kowalevskii
Comparative Biochemistry and Physiology, Part B 232 (2019) 87–92
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Cloning and characterization of a novel aspartate/glutamate racemase from the acorn worm Saccoglossus kowalevskii
T
⁎
Kouji Udaa, , Naoki Ishizukaa, Yumika Edashigea, Arika Kikuchia, Atanas D. Radkovb, Luke A. Moec a
Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520, Japan Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA c Department of Plant and Soil Sciences, 311 Plant Science Building, University of Kentucky, Lexington, KY 40546-0312, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: D-amino acid Aspartate racemase Glutamate racemase D-Glu D-asp
Previously, we demonstrated that the animal aspartate racemase (AspR) gene has evolved from the serine racemase (SerR) gene by acquisition of three consecutive serine residues (Ser155-Ser156-Ser157) involved in the strong AspR activity, and this event has occurred independently and frequently during animal evolution. In the present study, we cloned and characterized two mammalian SerR homologous genes from the hemichordate acorn worm (Saccoglossus kowalevskii). The enzymes have been identified as an AspR and an aspartate/glutamate racemase (Asp/GluR) on the basis of their kinetic parameters. The S. kowalevskii Asp/GluR shows comparable substrate affinity and high catalytic efficiency (kcat/Km) for both aspartate and glutamate and is the first reported enzyme from animals that can synthesize D-glutamate. Amino acid sequence alignment analysis and site-directed mutagenesis studies have revealed that the amino acid residue at position 156, which is serine in AspR and alanine in Asp/GluR, is associated with binding and recognition of glutamate and aspartate. Phylogenetic analysis suggests that the S. kowalevskii AspR gene has evolved from the SerR gene after the divergence of hemichordata and vertebrate lineages by acquisition of the three serine residues at position 155 to 157 as in the case of other animal AspR genes. Furthermore, the S. kowalevskii Asp/GluR gene is the result of AspR gene duplication and several amino acid substitutions including that of the 156th serine residue with alanine. The fact that SerR has acquired substrate specificity towards aspartate or glutamate raises the possibility that synthesis of other D-amino acids is carried out by enzymes evolved from SerR.
1. Introduction
central nervous system and acts as neurotransmitter (Miao et al., 2006; Patel et al., 2017). In addition, D-serine and D-aspartate were found in various non-mammalian eukaryotic phyla and probably involved several biological functions (Rosenberg and Ennor, 1961; Corrigan and Srinivasan, 1966; Saitoh et al., 2012). D-Serine and D-aspartate in animals are known to be synthesized by serine racemase (SerR) and aspartate racemase (AspR), respectively, which catalyze the interconversion of the L- and D-enantiomers. Serine racemase was first purified from rat brain (Wolosker et al., 1999b) and its cDNA was subsequently cloned from several mammals (De Miranda et al., 2000; Wolosker et al., 1999a). An aspartate racemase gene cloned from the bivalve mollusc Scapharca broughtonii showed a 44% overall amino acid sequence identity with mammalian SerR, indicating that animal SerR and AspR genes have evolved from a common ancestral gene (Abe et al., 2006; Uda et al., 2016).
For a long time, D-amino acids were thought to be limited to microorganisms, however, recent investigations show that several free Damino acids are widely distributed among many animals and have important roles in biological functions (Hamase et al., 2002; Radkov and Moe, 2014). In particular, the distribution and physiological functions of D-serine and D-aspartate have been studied in many works. A high level of free D-serine has been observed in mammalian brain where it acts as a positive modulators of signal transduction at the Nmethyl-D-aspartate (NMDA) receptor (Mothet et al., 2000). Free D-aspartate has been found in a wide variety of cells and tissues of mammals and plays physiological roles in regulating developmental processes, hormone secretion, and steroidogenesis (Katane and Homma, 2011). Also, in the marine mollusc Aplysia californica,D-aspartate exists in
Abbreviations: AspR, Aspartate racemase; Asp/GluR, Aspartate/glutamate racemase; BCA, Bicinchoninic Acid; PLP, Pyridoxal 5′-phosphate; SerR, Serine racemase ⁎ Corresponding author. E-mail address: [email protected] (K. Uda). https://doi.org/10.1016/j.cbpb.2019.03.006 Received 12 February 2019; Received in revised form 8 March 2019; Accepted 14 March 2019 Available online 19 March 2019 1096-4959/ © 2019 Elsevier Inc. All rights reserved.
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2.2. DNA synthesis and cloning of S. kowalevskii AspR and Asp/GluR genes
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 Dglutamate have been identified in animals (Ariyoshi et al., 2017; Katane et al., 2007, 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, 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 Dglutamate 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 Dglutamate in animals is synthesized by an amino acid 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.
The DNAs coding for S. kowalevskii AspR and Asp/GluR with a Cterminal 6 × His-tag sequence for convenience of purification by affinity chromatography were synthesized by the primer-extension PCR method as described previously (Uda et al., 2016). Primers for gene synthesis (Supplemental Table S1) were designed from S. kowalevskii AspR and Asp/GluR genes found in Genbank (accession no. XP_ 002735975 and XM_002735965, respectively) with the DNAWorks program (http://helixweb.nih.gov/dnaworks/). The open reading frames of the AspR and Asp/GluR genes were amplified and cloned into the pET30b vector (Novagen, WI, USA). The pET30b constructs were sequenced and confirmed that there were no unintended mutations in the coding region. 2.3. Construction of expression vectors of site-directed mutant and chimera proteins of S. kowalevskii AspR and Asp/GluR The pET30b constructs for S. kowalevskii AspR and Asp/GluR were used as templates for the mutagenesis. Site-directed mutagenesis on AspR and Asp/GluR gene was performed by inverse PCR as described previously (Suzuki et al., 2003). A series of chimera proteins between S. kowalevskii AspR and Asp/GluR were generated by overlap-extension PCR using 5′ or 3′ end gene specific primer and sense or antisense primers designed from the conserved amino acid sequence (KVRAV) at position 117 to 121. The cDNA insert was completely sequenced to confirm that only the intended mutations were introduced. The primers used for construction of site-directed mutant and chimera proteins were summarized in Supplemental Table S2. 2.4. Expression of S. kowalevskii AspR and asp/GluR genes in E. coli The recombinant genes were expressed in E. coli strain BL21 (DE3) cells by induction with 0.5 mM IPTG at 25 °C for 36 h. The E. coli cells were harvested by centrifugation and resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and were sonicated. The resultant soluble recombinant protein, after ultracentrifugation, was loaded onto a Ni-NTA Superflow column (QIAGEN, CA, USA) and the column was washed with wash buffer (50 mM NaH2PO4, 150 mM NaCl, 20 mM imidazole, pH 8.0). The purified protein was then eluted with elution buffer (50 mM NaH2PO4, 150 mM NaCl, 150 mM imidazole, pH 8.0) and the protein concentration of the purified enzymes was measured with the BCA-protein assay reagent (Thermo Fisher Scientific, MA, USA) with bovine serum albumin as a standard. All enzyme assays were conducted within 12 h of purification. 2.5. Enzyme assays The specific amino acid racemase activities of recombinant enzymes were measured at various concentrations of L and D isomers of aspartate and glutamate. The racemase assay and detection of resultant L- or Damino acids were performed as previously described (Uda et al., 2016). The reaction mixture contained 50 mM Tris/HCl (pH 8), 25 μM PLP, 1 mM DTT, 1 mM MgCl2, 1 mM ATP, 30 μL of purified recombinant enzyme, and a various concentration of substrate, in a final volume of 300 μL. Each condition was repeated at least three times independently and the negative control reactions were carried out in the absence of enzyme solution or with heat-inactivated enzymes. To determine the kinetic parameters, the data were fitted to Michaelis–Menten curves using SigmaPlot 12 (Systat Software, Inc.).
2. Materials and methods 2.1. Chemicals and materials L- and D-enantiomers of amino acids and N-tert-butyloxycarbonyl-Lcysteine were procured from Merck KGaA (Darmstadt, Germany). All other chemicals of regent grade were purchased from Merck KGaA (Darmstadt, Germany) or Wako (Osaka, Japan).
2.6. Alignment of amino acid sequences of AspR, Asp/GluR and SerR and construction of phylogenetic tree Multiple sequence alignment of AspRs, Asp/GluR and SerRs was generated by the MUSCLE program with default parameters (Edgar, 88
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agreement with that report, the phylogenetic tree generated in this study showed that S. kowalevskii AspR and Asp/GluR form a single clade, in which also fall closely related mammalian SerRs according to their taxonomic relationship (Fig. 1). Previously, on the basis of amino acid sequence alignment, we found conserved amino acid residues at position 155 to 157 (see Fig. 2, the amino acid residue numbers are based upon the S. kowalevskii AspR), which form a flexible loop and are closest to the substrate binding site in the crystal structure of Schizosaccharomyces pombe SerR (Uda et al., 2016). All known animal AspR enzymes have two or three serine residues at this region, while all SerR enzymes have no serine residues at the same positions. Thus, we referred to the two or three serine residues as the “triple serine loop region”, and we have shown that the triple serine loop region is necessary and sufficient for the strong AspR activity of serine/aspartate racemase family enzyme (Uda et al., 2017). It is notable that the Aplysia DAR1 was reported to have both serine and aspartate racemase activity (Wang et al., 2011), in spite of the fact that DAR1 has no serine residue at position 155 to 157 (Fig. 1). In addition, we have disclosed that mouse SerR also exhibits low but clearly detectable AspR activity using recombinant enzyme (Uda et al., 2016). These facts show that AspR activity is not only regulated by the triple serine loop region but also by other amino acid residues. However, the catalytic efficiency for L-aspartate of Aplysia DAR1 and mouse SerR are more than 400 times lower than that of Penaeus monodon, Crassostrea gigas and Acropora millepora AspRs, suggesting that the triple serine region is essential for strong AspR activity (Uda et al., 2016; Wang et al., 2011). The triple serine region is also present in S. kowalevskii AspR and Asp/GluR in accord with their AspR activity (Fig. 1). On the basis of these findings, the evolution of S. kowalevskii AspR and Asp/GluR genes was thought to have occurred as follows; first S. kowalevskii AspR gene has evolved from the ancestral SerR gene by acquisition of the triple serine loop region at its recent ancestral species, then Asp/GluR has arisen by gene duplication, which was followed by an increase of GluR activity.
2004). The maximum likelihood (ML) tree with the best-fit model (LG + G) was constructed using MEGA 7 (Tamura et al., 2013). 3. Results and discussion 3.1. Characterization of S. kowalevskii AspR and Asp/GluR The DNA sequences of two novel mammalian SerR homologs from S. kowalevskii were synthesized using a primer-extension PCR method to give the target amino acid sequences, which were expressed in E. coli as a His-tagged protein. We measured the amino acid racemase activities of S. kowalevskii recombinant enzymes using 10 mM of various amino acids. The S. kowalevskii enzymes showed significant activity only towards aspartate and glutamate and weak activity towards serine (Table 1) but showed no activity towards the other amino acids (alanine, arginine, asparagine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine). Several control experiments were conducted to confirm whether the GluR activity detected in this study was actually due to an enzymatic reaction (Supplemental Fig. S1) and indicated that D-glutamate was detected only when active S. kowalevskii enzyme was present. The kinetic parameters, Km and kcat values of the S. kowalevskii enzymes were determined by using seven different substrate concentrations (Table 2). One of the S. kowalevskii enzymes was identified as an AspR on the basis of its substrate specificity and kinetic properties. The other enzyme had comparable substrate affinity and catalytic efficiency (kcat/ Km) for aspartate and glutamate, indicating that this enzyme should be considered as an Asp/GluR. Compared with mouse SerR and Aplysia californica D-amino acid racemase 1 (DAR1) which synthesize D-serine and D-aspartate in vivo, respectively, the Km value for L-glutamate of S. kowalevskii Asp/GluR (23.0 mM) is 1.5–2.8 times higher than that for Lserine of mouse SerR (14.9 mM) and for L-aspartate of Aplysia DAR1 (8 mM) (Uda et al., 2016; Wang et al., 2011). In contrast, kcat value for −1 L-glutamate of S. kowalevskii Asp/GluR (6.70 s ) is more than 10 times higher than that for L-serine of mouse SerR (0.668 s−1) and for L-aspartate of Aplysia DAR1 (0.0321 s−1), and as a result, catalytic efficiency of S. kowalevskii Asp/GluR is more than 6 timers higher than that of mouse SerR and Aplysia DAR1 (Uda et al., 2016; Wang et al., 2011). This fact suggests that it is possible for the S. kowalevskii Asp/GluR to function in D-glutamate synthesis in vivo.
3.3. Key residues involved in glutamate racemase activity of S. kowalevskii Asp/GluR Despite high (85%) amino acid sequence identity (Fig. 2), there was more than 19-fold difference in specific activity for glutamate between S. kowalevskii AspR and Asp/GluR (Table 1). We carefully scrutinized the amino acid sequence alignment of animal AspRs, SerRs and the Asp/GluR from S. kowalevskii to identify which amino acids were responsible for GluR activity, and we found an amino acid residue at position 156 which is located in the triple serine loop region. The alanine residue is found at position 156 in S. kowalevskii Asp/GluR, while the residue P156 and S156 are strictly conserved in SerRs and AspRs, respectively (Fig. 1). The importance of residue 156 for substrate recognition has already been shown by site-directed mutagenesis in our recent study, in which the P156S mutant enzyme of mouse SerR increased AspR activities by approximately 20-fold compared to the wildtype variant (Uda et al., 2017). To verify the involvement of residue
3.2. Evolution of S. kowalevskii AspR and Asp/GluR Our previous phylogenetic analysis revealed three findings: (1) animal SerRs and AspRs are not separated by their specific racemase activity but separated according to their taxonomic group; (2) animal SerRs and AspRs form a serine/aspartate racemase family cluster and have evolved from a common ancestral gene; (3) Acropora millepora (Cnidaria), Crassostrea gigas (Mollusca) and Penaeus monodon (Arthropoda) AspRs have evolved from SerR independently by gene duplication at each recent ancestral species (Uda et al., 2016). In
Table 1 Specific activities (μmol/mg/h) of wild-type and mutant proteins of S. kowalevskii AspR and Asp/GluR. Reaction
L-Asp
to D-Asp to L-Asp L-Glu to D-Glu D-Glu to L-Glu L-Ser to D-Ser D-Ser to L-Ser D-Asp
Asp/GluR
AspR
wild-type
A156S
wild-type
S156A
145 ± 10 240 ± 36 195 ± 27 227 ± 18 1.02 ± 0.03 0.95 ± 0.10
459 ± 38 649 ± 64 254 ± 9 242 ± 24 4.5 ± 0.04 2.9 ± 0.06
2810 ± 340 4180 ± 440 10.3 ± 0.7 9.42 ± 0.25 1.22 ± 0.16 1.77 ± 0.22
563 ± 52 1120 ± 50 ND ND ND ND
These v [μmol/mg/h] values were obtained in the presence of 10 mM amino acids. ND: Not detected. 89
chimera 1
chimera 2
chimera 3
chimera 4
683 ± 13 513 ± 22 ND ND ND ND
49.2 ± 4.5 110 ± 6 ND ND ND ND
16.0 ± 1.4 18.8 ± 0.7 0.243 ± 0.007 0.345 ± 0.001 ND ND
480 ± 26 464 ± 70 1.19 ± 0.07 0.785 ± 0.068 ND ND
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Table 2 Comparison of kinetic constants of wild-type and mutant proteins of S. kowalevskii AspR and Asp/GluR. Enzyme (155th–157th residues)
Reaction
Km
L-Asp
to D-Asp to L-Asp L-Glu to D-Glu D-Glu to L-Glu L-Asp to D-Asp D-Asp to L-Asp L-Glu to D-Glu D-Glu to L-Glu L-Asp to D-Asp D-Asp to L-Asp L-Glu to D-Glu D-Glu to L-Glu L-Asp to D-Asp D-Asp to L-Asp L-Asp to D-Asp D-Asp to L-Asp L-Asp to D-Asp D-Asp to L-Asp L-Asp to D-Asp D-Asp to L-Asp L-Asp to D-Asp D-Asp to L-Asp
Asp/GluR wild-type (SAS)
D-Asp
Asp/GluR A156S (SSS)
AspR wild-type (SSS)
AspR S156A (SAS) chimera 1 (SSS) chimera 2 (SAS) chimera 3 (SAS) chimera 4 (SSS)
kcat −1
kcat/Km
[mM]
[s
]
15.4 ± 1.6 23.3 ± 4.9 23.0 ± 2.2 39.8 ± 3.9 3.27 ± 0.35 5.00 ± 0.74 101 ± 7 85.0 ± 13.4 3.52 ± 0.58 4.36 ± 0.59 107 ± 11 105 ± 11 10.4 ± 2.4 7.49 ± 0.79 3.63 ± 0.51 2.65 ± 0.28 6.07 ± 0.71 9.56 ± 0.87 17.6 ± 2.4 17.2 ± 3.6 13.6 ± 1.9 19.0 ± 3.1
3.63 ± 0.13 7.47 ± 0.64 6.70 ± 0.26 11.3 ± 0.7 6.19 ± 0.18 10.0 ± 0.4 18.2 ± 0.7 15.6 ± 1.5 41.6 ± 1.7 45.1 ± 1.6 1.13 ± 0.06 1.15 ± 0.06 17.9 ± 1.3 19.9 ± 0.7 9.70 ± 0.48 6.61 ± 0.22 0.784 ± 0.038 2.19 ± 0.09 0.465 ± 0.037 0.482 ± 0.057 10.6 ± 0.8 11.9 ± 1.1
[s−1·mM−1] 0.236 ± 0.026 0.321 ± 0.073 0.291 ± 0.031 0.284 ± 0.033 1.89 ± 0.21 2.00 ± 0.31 0.180 ± 0.015 0.183 ± 0.034 11.8 ± 2.0 10.4 ± 1.5 0.0106 ± 0.0012 0.0110 ± 0.0013 1.72 ± 0.41 2.65 ± 0.29 2.67 ± 0.40 2.49 ± 0.27 0.129 ± 0.016 0.229 ± 0.023 0.0265 ± 0.0043 0.0280 ± 0.0067 0.781 ± 0.124 0.627 ± 0.118
Each kinetic measurement has been repeated at least three times. Average and standard deviation are shown.
affinity for L- and D-aspartate by 4.7-fold but decreased the affinity for Land D-glutamate by 2.1–4.3-fold as compared with the wild-type. As a result, the A156S mutant of S. kowalevskii Asp/GluR showed approximately 10-fold higher catalytic efficiency for L- and D-aspartate as compared with L- and D-glutamate (Table 2). Thus, we thought that A156 and S156 are significantly associated with binding and recognition for glutamate and aspartate, respectively, resulting in changing the Km value. Moreover, a serine residue at position 156 increases the kcat value of both AspR and GluR reactions compared to an alanine residue. In agreement with this hypothesis, the S156A mutant of S. kowalevskii AspR showed 1.7–3-fold increase in Km value and 2.3-fold decrease in kcat value for L- and D-aspartate (Table 2). The kinetic parameters of GluR activity for the S156A mutant could not be obtained due to the reduction of the GluR activity by replacement of the serine residue by an alanine residue (Table 1). These results suggest that the alanine residue at position 156 is necessary but not sufficient for full GluR activity, and there are additional amino acid residues involved in GluR activity. Previously, we showed that a shift in substrate specificity from serine to aspartate in animal SerRs is caused by introducing only three residues (Ser155, Ser156 and Ser157) and evolution from SerR to AspR could have easily occurred (Uda et al., 2017). On the other hand, the present study demonstrates that a shift in substrate specificity from aspartate to glutamate requires more complicated amino acid substitutions.
155th-157th residues
SerR Penaeus monodon
100 78
AspR Penaeus monodon
90
SerR Milnesium tardigradum SerR Capitella teleta
100
DAR1 Aplysia californica AspR Scapharca broughtonii
82
AspR Crassostrea gigas
72 67
SerR Crassostrea gigas AspR Acropora millepora
99
SerR Acropora millepora 100
98
SerR Mus musculus SerR Homo sapiens Asp/GluR Saccoglossus kowalevskii
81 100
AspR Saccoglossus kowalevskii SerR Schizosaccharomyces pombe
HPF SSS HPY PPY PPY SSS SSS PPY PSS PPF HPN HPN SAS SSS PPY
0.10
Fig. 1. Phylogenetic tree based on the amino acid sequence of animal Asp/ GluR, AspRs and SerRs. The ML tree was constructed using the MEGA (Tamura et al., 2013). The ML bootstrap values are shown at the branching point. The following sequences were used for alignment and phylogenetic tree; Penaeus monodon AspR (GenBank Accession Nos. LC041007) and SerR (LC041008), Crassostrea gigas AspR (EKC33218) and SerR (LC041009), Acropora millepora AspR (JT020910) and SerR (JT017883), Capitella teleta SerR (ELT90787), Scapharca broughtonii AspR (BAE78960), Homo sapiens SerR (NP_068766), Mus musculus SerR (NM_013761), Milnesium tardigradum SerR (EZ759262) and Schizosaccharomyces pombe SerR (NP_587715). The amino acid sequence at position 155 to 157 (the amino acid residue numbers are based upon the S. kowalevskii AspR) are shown to the right of each sequence name. S. kowalevskii AspR and Asp/ GluR are shown as underlined.
3.4. Analysis of chimeric fusion proteins of S. kowalevskii AspR and Asp/ GluR While aspartate and glutamate are similar amino acids, AspR strictly distinguishes between the two. To identify the location of the amino acid residues involved in recognizing aspartate and glutamate, we generated four chimeric fusion proteins (chimeras 1–4 in Fig. 3) combining the separated N-terminal region and the C-terminal region of the wild-type and above-mentioned mutant proteins of S. kowalevskii AspR and Asp/GluR. The N-terminal region and the C-terminal region were joined at positions 117–121 (K-V-R-A-V) which is conserved in S. kowalevskii AspR and Asp/GluR (Fig. 2). Unfortunately, all chimeric proteins almost lost the GluR activity and this result suggests that both
156 in GluR activity, we introduced the A156S and S156A mutation into S. kowalevskii Asp/GluR and AspR, respectively, in the present study. The A156S mutant of S. kowalevskii Asp/GluR showed 1.3–2.7fold increase in kcat values for both AspR and GluR activity compared with wild-type (Table 2). Whereas, the mutant enzyme increased the 90
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10 20 30 40 50 60 70 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|
Asp/GluR MSKENKYCISYEDVQKAVERIKPAAHVTPVMTCRTMDRFSGGRHLYFKCELFQKIGAFKYRGAYNAIRHL 70 AspR MSGEKKYCISYEDVQKAVERIKPAAHVTPVMTSQTMDRFSGGRHLYFKCELFQKIGAFKFRGAYNAISHL 70 80 90 100 110 120 130 140 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|
Asp/GluR IDDCPDKSKVEVVTHSSGNHGQAVSLTAKEAGIKAYIVMQNNTPDVKVRAVEGYGGTVILCEPSEKAREA 140 AspR IDDCPDKSKVKVVTHSSGNHGQALALAAKMAGVQAYIVMQNISPAIKVRAVKEYGATVVECEPGEKARED 140 150 160 170 180 190 200 210 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|
Asp/GluR VAQKIVEETGASFISASQHPDVIAGQATMAVELLNEVPNLDAIVAPVSGGGMVAGICIAAKHIKPDIKIY 210 AspR ATNKVMAETGAIFISSSQHPDVIAGQGTMAVELLNEVPNLDAIVAPVSGGGMVAGICIAAKHIKPDIKIY 210 220 230 240 250 260 270 280 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|
Asp/GluR AAEPINADDCAKSLAAGHIIPLPGPPDTIADGLRATVGALTFPIIKDHLEDVITVKEEEIKIATKLMWER 280 AspR AAEPINADDCAKSFAAGHRIPLPGPPDTIADGLKTSVGVNSFPIIKDNIEDVITVTEEEIKIATKLMWER 280 290 300 310 320 330 ....|....|....|....|....|....|....|....|....|....|....|.
Asp/GluR AKLCIEPSSGAAVAAVLSDKFKALPASIRNVGVILSGGNVDFSKMSALMSTVKCEI 336 AspR TKLCIEPSSGTAVAAVLSDKFKALPASIKNVGVILSGGNLDLSKLSDWF------- 329 Fig. 2. Alignment of the amino acid sequences of S. kowalevskii AspR and Asp/GluR. The triple serine loop region at amino acid position 155 to 157 is indicated by a solid box. The amino acid residues at position 117 to 121, in which the N-terminal and C-terminal region of the chimeric fusion proteins of S. kowalevskii AspR and Asp/GluR are joined, are indicated by a dotted box. Amino acids residues that differ between S. kowalevskii AspR and Asp/GluR are indicated by gray shading.
Km values (3.63–6.07 mM) and chimera 3, 4 and Asp/GluR share the high Km values (13.6–17.6 mM) (Fig. 3). This result suggests that the Cterminal region, in addition to amino acid residue 156 of S. kowalevskii AspR and Asp/GluR, contains a key residue involved in the recognition of aspartate. However, this result may be caused by the protein misfolding or subtle structural differences between chimeric protein and wild-type enzyme. Further study using substitution of amino acid residues in the C-terminal region of the wild-type enzyme is needed to clarify this point.
N-terminal and C-terminal regions of Asp/GluR contain the amino acid residues involved in the recognition of aspartate and glutamate, or that the chimeric proteins were misfolded. However, the chimeric protein analyses are available for further understanding the mechanism of AspR reaction by using their kinetic parameters (Table 2) and the graph in which the kcat is plotted against Km value (Fig. 3). Consistent with the result of the S156A mutant of S. kowalevskii AspR described above, a single amino acid substitution of Ser156 to Ala in all proteins (indicated by arrows in the Fig. 3) leaded to an increase of Km value and a decrease of kcat value for AspR activity without exception. The result again showed the importance of residue Ser156 alone on kcat and Km values in AspR activity. Comparing the Km value for L-aspartate of all chimeras and wild-type enzymes shows that chimera 1, 2 and AspR share the low
A
1
117-121
155-156-157
Asp/GluR 117-121
1
155-156-157
AspR
Asp/GluR A156S
329
1
117-121
329
S S S 1
117-121
155-156-157
117-121
117-121
1
336
155-156-157
336
Fig. 3. Effects for kinetic parameters of mutant proteins and chimeric fusion proteins of S. kowalevskii AspR and Asp/GluR. (A) Structure of the wild-type, mutant proteins and chimeric fusion proteins (chimera 1–4) of S. kowalevskii AspR and Asp/GluR. The gray box and the white box correspond to the amino acid sequence of S. kowalevskii AspR and Asp/GluR, respectively. The amino acid sequence at position 155 to 157 are shown in each box. (B) Graph of the kcatvs Km values for L-Asp of the wild-type and chimeric fusion proteins.
155-156-157
329
S A S 117-121
chimera 2
S A S
This study has determined the kinetic parameters of two S S S
AspR S156A
155-156-157
chimera 3
1
1
S S S
chimera 1
B
336
S A S
4. Conclusion
155-156-157
329
S A S 1
117-121
chimera 4
155-156-157
336
S S S
45
AspR 40
kcat [s-1]
20
AspR S156A
15
10
Asp/GluR A156S
5
0
chimera 4
chimera 1
Asp/GluR
chimera 2 0
2
4
6
8
10
12
14
chimera 3 16
Km [mM] 91
18
20
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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 Dglutamate 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 Dglutamate 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. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbpb.2019.03.006.
encoding mouse D-aspartate oxidase and functional characterization of its recombinant proteins by site-directed mutagenesis. Amino Acids 32, 69–78. Katane, M., Saitoh, Y., Seida, Y., Sekine, M., Furuchi, T., Homma, H., 2010. Comparative characterization of three D-aspartate oxidases and one D-amino acid oxidase from Caenorhabditis elegans. Chem. Biodivers. 7, 1424–1434. Katane, M., Saitoh, Y., Uchiyama, K., Nakayama, K., Saitoh, Y., Miyamoto, T., Sekine, M., Uda, K., Homma, H., 2016. Characterization of a homologue of mammalian serine racemase from Caenorhabditis elegans: the enzyme is not critical for the metabolism of serine in vivo. Genes Cells 21, 966–977. Kera, Y., Aoyama, H., Matsumura, H., Hasegawa, A., Nagasaki, H., Yamada, R., 1995. Presence of free D-glutamate and D-aspartate in rat tissues. Biochim. Biophys. Acta 1243, 283–286. Kera, Y., Watanabe, T., Shibata, K., Nagasaki, H., Yamada, R.-h., 2001. D-aspartate oxidase and free acidic D-amino acids in lower vertebrates. J. Mol. Catal. B Enzym. 12, 121–129. Mangas, A., Covenas, R., Bodet, D., Geffard, M., Aguilar, L.A., Yajeya, J., 2007. Immunocytochemical visualization of D-glutamate in the rat brain. Neuroscience 144, 654–664. Meister, A., Bukenberger, M.W., Strassburger, M., 1963. The optically-specific enzymatic cyclization of D-glutamate. Biochem. Z. 338, 217–229. Miao, H., Rubakhin, S.S., Scanlan, C.R., Wang, L., Sweedler, J.V., 2006. D-Aspartate as a putative cell-cell signaling molecule in the Aplysia californica central nervous system. J. Neurochem. 97, 595–606. Mothet, J.P., Parent, A.T., Wolosker, H., Brady Jr., R.O., Linden, D.J., Ferris, C.D., Rogawski, M.A., Snyder, S.H., 2000. D-serine is an endogenous ligand for the glycine site of the N-methyl-D-aspartate receptor. Proc. Natl. Acad. Sci. U. S. A. 97, 4926–4931. Pan, Z.Z., Tong, G., Jahr, C.E., 1993. A false transmitter at excitatory synapses. Neuron 11, 85–91. Patel, A.V., Kawai, T., Wang, L., Rubakhin, S.S., Sweedler, J.V., 2017. Chiral measurement of aspartate and glutamate in single neurons by large-volume sample stacking capillary electrophoresis. Anal. Chem. 89, 12375–12382. Radkov, A.D., Moe, L.A., 2014. Bacterial synthesis of D-amino acids. Appl. Microbiol. Biotechnol. 98, 5363–5374. Rocca, E., Ghiretti, F., 1958. Purification and properties of D-glutamic acid oxidase from Octopus vulgaris lam. Arch. Biochem. Biophys. 77, 336–349. Rosenberg, H., Ennor, A.H., 1961. The occurrence of free D-serine in the earthworm. Biochem. J. 79, 424–428. Saitoh, Y., Katane, M., Kawata, T., Maeda, K., Sekine, M., Furuchi, T., Kobuna, H., Sakamoto, T., Inoue, T., Arai, H., Nakagawa, Y., Homma, H., 2012. Spatiotemporal localization of D-amino acid oxidase and D-aspartate oxidases during development in Caenorhabditis elegans. Mol. Cell. Biol. 32, 1967–1983. Sarower, M.G., Matsui, T., Abe, H., 2004. Distribution and substrate specificity of D-amino acid and D-aspartate oxidases in marine invertebrates. Sci. Asia 30, 335–340. Sekimizu, K., Larranaga, J., Hamamoto, H., Sekine, M., Furuchi, T., Katane, M., Homma, H., Matsuki, N., 2005. D-glutamic acid-induced muscle contraction in the silkworm, Bombyx mori. J. Biochem. 137, 199. Simakov, O., Kawashima, T., Marlétaz, F., Jenkins, J., Koyanagi, R., Mitros, T., Hisata, K., Bredeson, J., Shoguchi, E., Gyoja, F., 2015. Hemichordate genomes and deuterostome origins. Nature 527, 459. Suzuki, T., Taniguchi, T., Uda, K., 2003. Kinetic properties and structural characteristics of an unusual two-domain arginine kinase of the clam Corbicula japonica. FEBS Lett. 33, 95–98. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. evol. 30, 2725–2729. Tarui, A., Shibata, K., Takahashi, S., Kera, Y., Munegumi, T., Yamada, R.H., 2003. Nmethyl-D-glutamate and N-methyl-L-glutamate in Scapharca broughtonii (Mollusca) and other invertebrates. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 134, 79–87. Uda, K., Abe, K., Dehara, Y., Mizobata, K., Sogawa, N., Akagi, Y., Saigan, M., Radkov, A.D., Moe, L.A., 2016. Distribution and evolution of the serine/aspartate racemase family in invertebrates. Amino Acids 48, 387–402. Uda, K., Abe, K., Dehara, Y., Mizobata, K., Edashige, Y., Nishimura, R., Radkov, A.D., Moe, L.A., 2017. Triple serine loop region regulates the aspartate racemase activity of the serine/aspartate racemase family. 9Amino Acids4 49, 1743–1754. Wang, L., Ota, N., Romanova, E.V., Sweedler, J.V., 2011. A novel pyridoxal 5′-phosphatedependent amino acid racemase in the Aplysia californica central nervous system. J. Biol. Chem. 286, 13765–13774. Wolosker, H., Blackshaw, S., Snyder, S.H., 1999a. Serine racemase: a glial enzyme synthesizing D-serine to regulate glutamate-N-methyl-D-aspartate neurotransmission. Proc. Natl. Acad. Sci. U. S. A. 96, 13409–13414. Wolosker, H., Sheth, K.N., Takahashi, M., Mothet, J.P., Brady Jr., R.O., Ferris, C.D., Snyder, S.H., 1999b. Purification of serine racemase: biosynthesis of the neuromodulator D-serine. Proc. Natl. Acad. Sci. U. S. A. 96, 721–725. Yoshikawa, N., Ashida, W., Hamase, K., Abe, H., 2011. HPLC determination of the distribution of D-amino acids and effects of ecdysis on alanine racemase activity in kuruma prawn Marsupenaeus japonicus. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 879, 3283–3288. Zaar, K., Kost, H.P., Schad, A., Volkl, A., Baumgart, E., Fahimi, H.D., 2002. Cellular and subcellular distribution of D-aspartate oxidase in human and rat brain. J. Comp. Neurol. 450, 272–282.
Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by JSPS KAKENHI Grant Numbers 24770068 and 15K07152. References Abe, K., Takahashi, S., Muroki, Y., Kera, Y., Yamada, R.-h., 2006. Cloning and expression of the Pyridoxal 5′-phosphate–dependent aspartate Racemase gene from the bivalve Mollusk Scapharca broughtonii and characterization of the recombinant enzyme. J. Biochem. 139, 235–244. Ariyoshi, M., Katane, M., Hamase, K., Miyoshi, Y., Nakane, M., Hoshino, A., Okawa, Y., Mita, Y., Kaimoto, S., Uchihashi, M., Fukai, K., Ono, K., Tateishi, S., Hato, D., Yamanaka, R., Honda, S., Fushimura, Y., Iwai-Kanai, E., Ishihara, N., Mita, M., Homma, H., Matoba, S., 2017. D-glutamate is metabolized in the heart mitochondria. Sci. Rep. 7, 43911. Corrigan, J.J., 1969. D-amino acids in animals. Science 164, 142–149. Corrigan, J.J., Srinivasan, N.G., 1966. The occurrence of certain D-amino acids in insects. Biochemistry 5, 1185–1190. De Miranda, J., Santoro, A., Engelender, S., Wolosker, H., 2000. Human serine racemase: molecular cloning, genomic organization and functional analysis. Gene 256, 183–188. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Freeman Jr., R., Wu, M., Cordonnier-Pratt, M., Pratt, L., Gruber, C., Smith, M., Lander, E., Stange-Thomann, N., Lowe, C., Gerhart, J., 2008. cDNA sequences for transcription factors and signaling proteins of the hemichordate Saccoglossus kowalevskii: efficacy of the expressed sequence tag (EST) approach for evolutionary and developmental studies of a new organism. Biol. Bull. 214, 284. Hamase, K., Morikawa, A., Zaitsu, K., 2002. D-amino acids in mammals and their diagnostic value. J. Chromatogr. B 781, 73–91. Han, H., Miyoshi, Y., Ueno, K., Okamura, C., Tojo, Y., Mita, M., Lindner, W., Zaitsu, K., Hamase, K., 2011. Simultaneous determination of D-aspartic acid and D-glutamic acid in rat tissues and physiological fluids using a multi-loop two-dimensional HPLC procedure. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 879, 3196. Katane, M., Homma, H., 2011. D-aspartate–an important bioactive substance in mammals: a review from an analytical and biological point of view. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 879, 3108–3121. Katane, M., Furuchi, T., Sekine, M., Homma, H., 2007. Molecular cloning of a cDNA
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Cloning and characterization of a novel aspartate/glutamate racemase from the acorn worm Saccoglossus kowalevskii
T
⁎
Kouji Udaa, , Naoki Ishizukaa, Yumika Edashigea, Arika Kikuchia, Atanas D. Radkovb, Luke A. Moec a
Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520, Japan Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA c Department of Plant and Soil Sciences, 311 Plant Science Building, University of Kentucky, Lexington, KY 40546-0312, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: D-amino acid Aspartate racemase Glutamate racemase D-Glu D-asp
Previously, we demonstrated that the animal aspartate racemase (AspR) gene has evolved from the serine racemase (SerR) gene by acquisition of three consecutive serine residues (Ser155-Ser156-Ser157) involved in the strong AspR activity, and this event has occurred independently and frequently during animal evolution. In the present study, we cloned and characterized two mammalian SerR homologous genes from the hemichordate acorn worm (Saccoglossus kowalevskii). The enzymes have been identified as an AspR and an aspartate/glutamate racemase (Asp/GluR) on the basis of their kinetic parameters. The S. kowalevskii Asp/GluR shows comparable substrate affinity and high catalytic efficiency (kcat/Km) for both aspartate and glutamate and is the first reported enzyme from animals that can synthesize D-glutamate. Amino acid sequence alignment analysis and site-directed mutagenesis studies have revealed that the amino acid residue at position 156, which is serine in AspR and alanine in Asp/GluR, is associated with binding and recognition of glutamate and aspartate. Phylogenetic analysis suggests that the S. kowalevskii AspR gene has evolved from the SerR gene after the divergence of hemichordata and vertebrate lineages by acquisition of the three serine residues at position 155 to 157 as in the case of other animal AspR genes. Furthermore, the S. kowalevskii Asp/GluR gene is the result of AspR gene duplication and several amino acid substitutions including that of the 156th serine residue with alanine. The fact that SerR has acquired substrate specificity towards aspartate or glutamate raises the possibility that synthesis of other D-amino acids is carried out by enzymes evolved from SerR.
1. Introduction
central nervous system and acts as neurotransmitter (Miao et al., 2006; Patel et al., 2017). In addition, D-serine and D-aspartate were found in various non-mammalian eukaryotic phyla and probably involved several biological functions (Rosenberg and Ennor, 1961; Corrigan and Srinivasan, 1966; Saitoh et al., 2012). D-Serine and D-aspartate in animals are known to be synthesized by serine racemase (SerR) and aspartate racemase (AspR), respectively, which catalyze the interconversion of the L- and D-enantiomers. Serine racemase was first purified from rat brain (Wolosker et al., 1999b) and its cDNA was subsequently cloned from several mammals (De Miranda et al., 2000; Wolosker et al., 1999a). An aspartate racemase gene cloned from the bivalve mollusc Scapharca broughtonii showed a 44% overall amino acid sequence identity with mammalian SerR, indicating that animal SerR and AspR genes have evolved from a common ancestral gene (Abe et al., 2006; Uda et al., 2016).
For a long time, D-amino acids were thought to be limited to microorganisms, however, recent investigations show that several free Damino acids are widely distributed among many animals and have important roles in biological functions (Hamase et al., 2002; Radkov and Moe, 2014). In particular, the distribution and physiological functions of D-serine and D-aspartate have been studied in many works. A high level of free D-serine has been observed in mammalian brain where it acts as a positive modulators of signal transduction at the Nmethyl-D-aspartate (NMDA) receptor (Mothet et al., 2000). Free D-aspartate has been found in a wide variety of cells and tissues of mammals and plays physiological roles in regulating developmental processes, hormone secretion, and steroidogenesis (Katane and Homma, 2011). Also, in the marine mollusc Aplysia californica,D-aspartate exists in
Abbreviations: AspR, Aspartate racemase; Asp/GluR, Aspartate/glutamate racemase; BCA, Bicinchoninic Acid; PLP, Pyridoxal 5′-phosphate; SerR, Serine racemase ⁎ Corresponding author. E-mail address: [email protected] (K. Uda). https://doi.org/10.1016/j.cbpb.2019.03.006 Received 12 February 2019; Received in revised form 8 March 2019; Accepted 14 March 2019 Available online 19 March 2019 1096-4959/ © 2019 Elsevier Inc. All rights reserved.
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2.2. DNA synthesis and cloning of S. kowalevskii AspR and Asp/GluR genes
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 Dglutamate have been identified in animals (Ariyoshi et al., 2017; Katane et al., 2007, 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, 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 Dglutamate 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 Dglutamate in animals is synthesized by an amino acid 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.
The DNAs coding for S. kowalevskii AspR and Asp/GluR with a Cterminal 6 × His-tag sequence for convenience of purification by affinity chromatography were synthesized by the primer-extension PCR method as described previously (Uda et al., 2016). Primers for gene synthesis (Supplemental Table S1) were designed from S. kowalevskii AspR and Asp/GluR genes found in Genbank (accession no. XP_ 002735975 and XM_002735965, respectively) with the DNAWorks program (http://helixweb.nih.gov/dnaworks/). The open reading frames of the AspR and Asp/GluR genes were amplified and cloned into the pET30b vector (Novagen, WI, USA). The pET30b constructs were sequenced and confirmed that there were no unintended mutations in the coding region. 2.3. Construction of expression vectors of site-directed mutant and chimera proteins of S. kowalevskii AspR and Asp/GluR The pET30b constructs for S. kowalevskii AspR and Asp/GluR were used as templates for the mutagenesis. Site-directed mutagenesis on AspR and Asp/GluR gene was performed by inverse PCR as described previously (Suzuki et al., 2003). A series of chimera proteins between S. kowalevskii AspR and Asp/GluR were generated by overlap-extension PCR using 5′ or 3′ end gene specific primer and sense or antisense primers designed from the conserved amino acid sequence (KVRAV) at position 117 to 121. The cDNA insert was completely sequenced to confirm that only the intended mutations were introduced. The primers used for construction of site-directed mutant and chimera proteins were summarized in Supplemental Table S2. 2.4. Expression of S. kowalevskii AspR and asp/GluR genes in E. coli The recombinant genes were expressed in E. coli strain BL21 (DE3) cells by induction with 0.5 mM IPTG at 25 °C for 36 h. The E. coli cells were harvested by centrifugation and resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and were sonicated. The resultant soluble recombinant protein, after ultracentrifugation, was loaded onto a Ni-NTA Superflow column (QIAGEN, CA, USA) and the column was washed with wash buffer (50 mM NaH2PO4, 150 mM NaCl, 20 mM imidazole, pH 8.0). The purified protein was then eluted with elution buffer (50 mM NaH2PO4, 150 mM NaCl, 150 mM imidazole, pH 8.0) and the protein concentration of the purified enzymes was measured with the BCA-protein assay reagent (Thermo Fisher Scientific, MA, USA) with bovine serum albumin as a standard. All enzyme assays were conducted within 12 h of purification. 2.5. Enzyme assays The specific amino acid racemase activities of recombinant enzymes were measured at various concentrations of L and D isomers of aspartate and glutamate. The racemase assay and detection of resultant L- or Damino acids were performed as previously described (Uda et al., 2016). The reaction mixture contained 50 mM Tris/HCl (pH 8), 25 μM PLP, 1 mM DTT, 1 mM MgCl2, 1 mM ATP, 30 μL of purified recombinant enzyme, and a various concentration of substrate, in a final volume of 300 μL. Each condition was repeated at least three times independently and the negative control reactions were carried out in the absence of enzyme solution or with heat-inactivated enzymes. To determine the kinetic parameters, the data were fitted to Michaelis–Menten curves using SigmaPlot 12 (Systat Software, Inc.).
2. Materials and methods 2.1. Chemicals and materials L- and D-enantiomers of amino acids and N-tert-butyloxycarbonyl-Lcysteine were procured from Merck KGaA (Darmstadt, Germany). All other chemicals of regent grade were purchased from Merck KGaA (Darmstadt, Germany) or Wako (Osaka, Japan).
2.6. Alignment of amino acid sequences of AspR, Asp/GluR and SerR and construction of phylogenetic tree Multiple sequence alignment of AspRs, Asp/GluR and SerRs was generated by the MUSCLE program with default parameters (Edgar, 88
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agreement with that report, the phylogenetic tree generated in this study showed that S. kowalevskii AspR and Asp/GluR form a single clade, in which also fall closely related mammalian SerRs according to their taxonomic relationship (Fig. 1). Previously, on the basis of amino acid sequence alignment, we found conserved amino acid residues at position 155 to 157 (see Fig. 2, the amino acid residue numbers are based upon the S. kowalevskii AspR), which form a flexible loop and are closest to the substrate binding site in the crystal structure of Schizosaccharomyces pombe SerR (Uda et al., 2016). All known animal AspR enzymes have two or three serine residues at this region, while all SerR enzymes have no serine residues at the same positions. Thus, we referred to the two or three serine residues as the “triple serine loop region”, and we have shown that the triple serine loop region is necessary and sufficient for the strong AspR activity of serine/aspartate racemase family enzyme (Uda et al., 2017). It is notable that the Aplysia DAR1 was reported to have both serine and aspartate racemase activity (Wang et al., 2011), in spite of the fact that DAR1 has no serine residue at position 155 to 157 (Fig. 1). In addition, we have disclosed that mouse SerR also exhibits low but clearly detectable AspR activity using recombinant enzyme (Uda et al., 2016). These facts show that AspR activity is not only regulated by the triple serine loop region but also by other amino acid residues. However, the catalytic efficiency for L-aspartate of Aplysia DAR1 and mouse SerR are more than 400 times lower than that of Penaeus monodon, Crassostrea gigas and Acropora millepora AspRs, suggesting that the triple serine region is essential for strong AspR activity (Uda et al., 2016; Wang et al., 2011). The triple serine region is also present in S. kowalevskii AspR and Asp/GluR in accord with their AspR activity (Fig. 1). On the basis of these findings, the evolution of S. kowalevskii AspR and Asp/GluR genes was thought to have occurred as follows; first S. kowalevskii AspR gene has evolved from the ancestral SerR gene by acquisition of the triple serine loop region at its recent ancestral species, then Asp/GluR has arisen by gene duplication, which was followed by an increase of GluR activity.
2004). The maximum likelihood (ML) tree with the best-fit model (LG + G) was constructed using MEGA 7 (Tamura et al., 2013). 3. Results and discussion 3.1. Characterization of S. kowalevskii AspR and Asp/GluR The DNA sequences of two novel mammalian SerR homologs from S. kowalevskii were synthesized using a primer-extension PCR method to give the target amino acid sequences, which were expressed in E. coli as a His-tagged protein. We measured the amino acid racemase activities of S. kowalevskii recombinant enzymes using 10 mM of various amino acids. The S. kowalevskii enzymes showed significant activity only towards aspartate and glutamate and weak activity towards serine (Table 1) but showed no activity towards the other amino acids (alanine, arginine, asparagine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine). Several control experiments were conducted to confirm whether the GluR activity detected in this study was actually due to an enzymatic reaction (Supplemental Fig. S1) and indicated that D-glutamate was detected only when active S. kowalevskii enzyme was present. The kinetic parameters, Km and kcat values of the S. kowalevskii enzymes were determined by using seven different substrate concentrations (Table 2). One of the S. kowalevskii enzymes was identified as an AspR on the basis of its substrate specificity and kinetic properties. The other enzyme had comparable substrate affinity and catalytic efficiency (kcat/ Km) for aspartate and glutamate, indicating that this enzyme should be considered as an Asp/GluR. Compared with mouse SerR and Aplysia californica D-amino acid racemase 1 (DAR1) which synthesize D-serine and D-aspartate in vivo, respectively, the Km value for L-glutamate of S. kowalevskii Asp/GluR (23.0 mM) is 1.5–2.8 times higher than that for Lserine of mouse SerR (14.9 mM) and for L-aspartate of Aplysia DAR1 (8 mM) (Uda et al., 2016; Wang et al., 2011). In contrast, kcat value for −1 L-glutamate of S. kowalevskii Asp/GluR (6.70 s ) is more than 10 times higher than that for L-serine of mouse SerR (0.668 s−1) and for L-aspartate of Aplysia DAR1 (0.0321 s−1), and as a result, catalytic efficiency of S. kowalevskii Asp/GluR is more than 6 timers higher than that of mouse SerR and Aplysia DAR1 (Uda et al., 2016; Wang et al., 2011). This fact suggests that it is possible for the S. kowalevskii Asp/GluR to function in D-glutamate synthesis in vivo.
3.3. Key residues involved in glutamate racemase activity of S. kowalevskii Asp/GluR Despite high (85%) amino acid sequence identity (Fig. 2), there was more than 19-fold difference in specific activity for glutamate between S. kowalevskii AspR and Asp/GluR (Table 1). We carefully scrutinized the amino acid sequence alignment of animal AspRs, SerRs and the Asp/GluR from S. kowalevskii to identify which amino acids were responsible for GluR activity, and we found an amino acid residue at position 156 which is located in the triple serine loop region. The alanine residue is found at position 156 in S. kowalevskii Asp/GluR, while the residue P156 and S156 are strictly conserved in SerRs and AspRs, respectively (Fig. 1). The importance of residue 156 for substrate recognition has already been shown by site-directed mutagenesis in our recent study, in which the P156S mutant enzyme of mouse SerR increased AspR activities by approximately 20-fold compared to the wildtype variant (Uda et al., 2017). To verify the involvement of residue
3.2. Evolution of S. kowalevskii AspR and Asp/GluR Our previous phylogenetic analysis revealed three findings: (1) animal SerRs and AspRs are not separated by their specific racemase activity but separated according to their taxonomic group; (2) animal SerRs and AspRs form a serine/aspartate racemase family cluster and have evolved from a common ancestral gene; (3) Acropora millepora (Cnidaria), Crassostrea gigas (Mollusca) and Penaeus monodon (Arthropoda) AspRs have evolved from SerR independently by gene duplication at each recent ancestral species (Uda et al., 2016). In
Table 1 Specific activities (μmol/mg/h) of wild-type and mutant proteins of S. kowalevskii AspR and Asp/GluR. Reaction
L-Asp
to D-Asp to L-Asp L-Glu to D-Glu D-Glu to L-Glu L-Ser to D-Ser D-Ser to L-Ser D-Asp
Asp/GluR
AspR
wild-type
A156S
wild-type
S156A
145 ± 10 240 ± 36 195 ± 27 227 ± 18 1.02 ± 0.03 0.95 ± 0.10
459 ± 38 649 ± 64 254 ± 9 242 ± 24 4.5 ± 0.04 2.9 ± 0.06
2810 ± 340 4180 ± 440 10.3 ± 0.7 9.42 ± 0.25 1.22 ± 0.16 1.77 ± 0.22
563 ± 52 1120 ± 50 ND ND ND ND
These v [μmol/mg/h] values were obtained in the presence of 10 mM amino acids. ND: Not detected. 89
chimera 1
chimera 2
chimera 3
chimera 4
683 ± 13 513 ± 22 ND ND ND ND
49.2 ± 4.5 110 ± 6 ND ND ND ND
16.0 ± 1.4 18.8 ± 0.7 0.243 ± 0.007 0.345 ± 0.001 ND ND
480 ± 26 464 ± 70 1.19 ± 0.07 0.785 ± 0.068 ND ND
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Table 2 Comparison of kinetic constants of wild-type and mutant proteins of S. kowalevskii AspR and Asp/GluR. Enzyme (155th–157th residues)
Reaction
Km
L-Asp
to D-Asp to L-Asp L-Glu to D-Glu D-Glu to L-Glu L-Asp to D-Asp D-Asp to L-Asp L-Glu to D-Glu D-Glu to L-Glu L-Asp to D-Asp D-Asp to L-Asp L-Glu to D-Glu D-Glu to L-Glu L-Asp to D-Asp D-Asp to L-Asp L-Asp to D-Asp D-Asp to L-Asp L-Asp to D-Asp D-Asp to L-Asp L-Asp to D-Asp D-Asp to L-Asp L-Asp to D-Asp D-Asp to L-Asp
Asp/GluR wild-type (SAS)
D-Asp
Asp/GluR A156S (SSS)
AspR wild-type (SSS)
AspR S156A (SAS) chimera 1 (SSS) chimera 2 (SAS) chimera 3 (SAS) chimera 4 (SSS)
kcat −1
kcat/Km
[mM]
[s
]
15.4 ± 1.6 23.3 ± 4.9 23.0 ± 2.2 39.8 ± 3.9 3.27 ± 0.35 5.00 ± 0.74 101 ± 7 85.0 ± 13.4 3.52 ± 0.58 4.36 ± 0.59 107 ± 11 105 ± 11 10.4 ± 2.4 7.49 ± 0.79 3.63 ± 0.51 2.65 ± 0.28 6.07 ± 0.71 9.56 ± 0.87 17.6 ± 2.4 17.2 ± 3.6 13.6 ± 1.9 19.0 ± 3.1
3.63 ± 0.13 7.47 ± 0.64 6.70 ± 0.26 11.3 ± 0.7 6.19 ± 0.18 10.0 ± 0.4 18.2 ± 0.7 15.6 ± 1.5 41.6 ± 1.7 45.1 ± 1.6 1.13 ± 0.06 1.15 ± 0.06 17.9 ± 1.3 19.9 ± 0.7 9.70 ± 0.48 6.61 ± 0.22 0.784 ± 0.038 2.19 ± 0.09 0.465 ± 0.037 0.482 ± 0.057 10.6 ± 0.8 11.9 ± 1.1
[s−1·mM−1] 0.236 ± 0.026 0.321 ± 0.073 0.291 ± 0.031 0.284 ± 0.033 1.89 ± 0.21 2.00 ± 0.31 0.180 ± 0.015 0.183 ± 0.034 11.8 ± 2.0 10.4 ± 1.5 0.0106 ± 0.0012 0.0110 ± 0.0013 1.72 ± 0.41 2.65 ± 0.29 2.67 ± 0.40 2.49 ± 0.27 0.129 ± 0.016 0.229 ± 0.023 0.0265 ± 0.0043 0.0280 ± 0.0067 0.781 ± 0.124 0.627 ± 0.118
Each kinetic measurement has been repeated at least three times. Average and standard deviation are shown.
affinity for L- and D-aspartate by 4.7-fold but decreased the affinity for Land D-glutamate by 2.1–4.3-fold as compared with the wild-type. As a result, the A156S mutant of S. kowalevskii Asp/GluR showed approximately 10-fold higher catalytic efficiency for L- and D-aspartate as compared with L- and D-glutamate (Table 2). Thus, we thought that A156 and S156 are significantly associated with binding and recognition for glutamate and aspartate, respectively, resulting in changing the Km value. Moreover, a serine residue at position 156 increases the kcat value of both AspR and GluR reactions compared to an alanine residue. In agreement with this hypothesis, the S156A mutant of S. kowalevskii AspR showed 1.7–3-fold increase in Km value and 2.3-fold decrease in kcat value for L- and D-aspartate (Table 2). The kinetic parameters of GluR activity for the S156A mutant could not be obtained due to the reduction of the GluR activity by replacement of the serine residue by an alanine residue (Table 1). These results suggest that the alanine residue at position 156 is necessary but not sufficient for full GluR activity, and there are additional amino acid residues involved in GluR activity. Previously, we showed that a shift in substrate specificity from serine to aspartate in animal SerRs is caused by introducing only three residues (Ser155, Ser156 and Ser157) and evolution from SerR to AspR could have easily occurred (Uda et al., 2017). On the other hand, the present study demonstrates that a shift in substrate specificity from aspartate to glutamate requires more complicated amino acid substitutions.
155th-157th residues
SerR Penaeus monodon
100 78
AspR Penaeus monodon
90
SerR Milnesium tardigradum SerR Capitella teleta
100
DAR1 Aplysia californica AspR Scapharca broughtonii
82
AspR Crassostrea gigas
72 67
SerR Crassostrea gigas AspR Acropora millepora
99
SerR Acropora millepora 100
98
SerR Mus musculus SerR Homo sapiens Asp/GluR Saccoglossus kowalevskii
81 100
AspR Saccoglossus kowalevskii SerR Schizosaccharomyces pombe
HPF SSS HPY PPY PPY SSS SSS PPY PSS PPF HPN HPN SAS SSS PPY
0.10
Fig. 1. Phylogenetic tree based on the amino acid sequence of animal Asp/ GluR, AspRs and SerRs. The ML tree was constructed using the MEGA (Tamura et al., 2013). The ML bootstrap values are shown at the branching point. The following sequences were used for alignment and phylogenetic tree; Penaeus monodon AspR (GenBank Accession Nos. LC041007) and SerR (LC041008), Crassostrea gigas AspR (EKC33218) and SerR (LC041009), Acropora millepora AspR (JT020910) and SerR (JT017883), Capitella teleta SerR (ELT90787), Scapharca broughtonii AspR (BAE78960), Homo sapiens SerR (NP_068766), Mus musculus SerR (NM_013761), Milnesium tardigradum SerR (EZ759262) and Schizosaccharomyces pombe SerR (NP_587715). The amino acid sequence at position 155 to 157 (the amino acid residue numbers are based upon the S. kowalevskii AspR) are shown to the right of each sequence name. S. kowalevskii AspR and Asp/ GluR are shown as underlined.
3.4. Analysis of chimeric fusion proteins of S. kowalevskii AspR and Asp/ GluR While aspartate and glutamate are similar amino acids, AspR strictly distinguishes between the two. To identify the location of the amino acid residues involved in recognizing aspartate and glutamate, we generated four chimeric fusion proteins (chimeras 1–4 in Fig. 3) combining the separated N-terminal region and the C-terminal region of the wild-type and above-mentioned mutant proteins of S. kowalevskii AspR and Asp/GluR. The N-terminal region and the C-terminal region were joined at positions 117–121 (K-V-R-A-V) which is conserved in S. kowalevskii AspR and Asp/GluR (Fig. 2). Unfortunately, all chimeric proteins almost lost the GluR activity and this result suggests that both
156 in GluR activity, we introduced the A156S and S156A mutation into S. kowalevskii Asp/GluR and AspR, respectively, in the present study. The A156S mutant of S. kowalevskii Asp/GluR showed 1.3–2.7fold increase in kcat values for both AspR and GluR activity compared with wild-type (Table 2). Whereas, the mutant enzyme increased the 90
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10 20 30 40 50 60 70 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|
Asp/GluR MSKENKYCISYEDVQKAVERIKPAAHVTPVMTCRTMDRFSGGRHLYFKCELFQKIGAFKYRGAYNAIRHL 70 AspR MSGEKKYCISYEDVQKAVERIKPAAHVTPVMTSQTMDRFSGGRHLYFKCELFQKIGAFKFRGAYNAISHL 70 80 90 100 110 120 130 140 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|
Asp/GluR IDDCPDKSKVEVVTHSSGNHGQAVSLTAKEAGIKAYIVMQNNTPDVKVRAVEGYGGTVILCEPSEKAREA 140 AspR IDDCPDKSKVKVVTHSSGNHGQALALAAKMAGVQAYIVMQNISPAIKVRAVKEYGATVVECEPGEKARED 140 150 160 170 180 190 200 210 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|
Asp/GluR VAQKIVEETGASFISASQHPDVIAGQATMAVELLNEVPNLDAIVAPVSGGGMVAGICIAAKHIKPDIKIY 210 AspR ATNKVMAETGAIFISSSQHPDVIAGQGTMAVELLNEVPNLDAIVAPVSGGGMVAGICIAAKHIKPDIKIY 210 220 230 240 250 260 270 280 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|
Asp/GluR AAEPINADDCAKSLAAGHIIPLPGPPDTIADGLRATVGALTFPIIKDHLEDVITVKEEEIKIATKLMWER 280 AspR AAEPINADDCAKSFAAGHRIPLPGPPDTIADGLKTSVGVNSFPIIKDNIEDVITVTEEEIKIATKLMWER 280 290 300 310 320 330 ....|....|....|....|....|....|....|....|....|....|....|.
Asp/GluR AKLCIEPSSGAAVAAVLSDKFKALPASIRNVGVILSGGNVDFSKMSALMSTVKCEI 336 AspR TKLCIEPSSGTAVAAVLSDKFKALPASIKNVGVILSGGNLDLSKLSDWF------- 329 Fig. 2. Alignment of the amino acid sequences of S. kowalevskii AspR and Asp/GluR. The triple serine loop region at amino acid position 155 to 157 is indicated by a solid box. The amino acid residues at position 117 to 121, in which the N-terminal and C-terminal region of the chimeric fusion proteins of S. kowalevskii AspR and Asp/GluR are joined, are indicated by a dotted box. Amino acids residues that differ between S. kowalevskii AspR and Asp/GluR are indicated by gray shading.
Km values (3.63–6.07 mM) and chimera 3, 4 and Asp/GluR share the high Km values (13.6–17.6 mM) (Fig. 3). This result suggests that the Cterminal region, in addition to amino acid residue 156 of S. kowalevskii AspR and Asp/GluR, contains a key residue involved in the recognition of aspartate. However, this result may be caused by the protein misfolding or subtle structural differences between chimeric protein and wild-type enzyme. Further study using substitution of amino acid residues in the C-terminal region of the wild-type enzyme is needed to clarify this point.
N-terminal and C-terminal regions of Asp/GluR contain the amino acid residues involved in the recognition of aspartate and glutamate, or that the chimeric proteins were misfolded. However, the chimeric protein analyses are available for further understanding the mechanism of AspR reaction by using their kinetic parameters (Table 2) and the graph in which the kcat is plotted against Km value (Fig. 3). Consistent with the result of the S156A mutant of S. kowalevskii AspR described above, a single amino acid substitution of Ser156 to Ala in all proteins (indicated by arrows in the Fig. 3) leaded to an increase of Km value and a decrease of kcat value for AspR activity without exception. The result again showed the importance of residue Ser156 alone on kcat and Km values in AspR activity. Comparing the Km value for L-aspartate of all chimeras and wild-type enzymes shows that chimera 1, 2 and AspR share the low
A
1
117-121
155-156-157
Asp/GluR 117-121
1
155-156-157
AspR
Asp/GluR A156S
329
1
117-121
329
S S S 1
117-121
155-156-157
117-121
117-121
1
336
155-156-157
336
Fig. 3. Effects for kinetic parameters of mutant proteins and chimeric fusion proteins of S. kowalevskii AspR and Asp/GluR. (A) Structure of the wild-type, mutant proteins and chimeric fusion proteins (chimera 1–4) of S. kowalevskii AspR and Asp/GluR. The gray box and the white box correspond to the amino acid sequence of S. kowalevskii AspR and Asp/GluR, respectively. The amino acid sequence at position 155 to 157 are shown in each box. (B) Graph of the kcatvs Km values for L-Asp of the wild-type and chimeric fusion proteins.
155-156-157
329
S A S 117-121
chimera 2
S A S
This study has determined the kinetic parameters of two S S S
AspR S156A
155-156-157
chimera 3
1
1
S S S
chimera 1
B
336
S A S
4. Conclusion
155-156-157
329
S A S 1
117-121
chimera 4
155-156-157
336
S S S
45
AspR 40
kcat [s-1]
20
AspR S156A
15
10
Asp/GluR A156S
5
0
chimera 4
chimera 1
Asp/GluR
chimera 2 0
2
4
6
8
10
12
14
chimera 3 16
Km [mM] 91
18
20
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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 Dglutamate 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 Dglutamate 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. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbpb.2019.03.006.
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