2R,3S - American Chemical Society

Article pubs.acs.org/JAFC

Occurrence of the (2R,3S)‑Isomer of 2‑Amino-3,4-dihydroxybutanoic Acid in the Mushroom Hypsizygus marmoreus Tomokazu Ito,† Zhuoer Yu,† Issei Yoshino,‡ Yurina Hirozawa,† Kana Yamamoto,† Kiyotugu Shinoda,§ Akira Watanabe,‡ Hisashi Hemmi,† Yasuhiko Asada,*,‡ and Tohru Yoshimura*,† †

Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furou-chou, Chikusa, Nagoya, Aichi 464-8601, Japan ‡ Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kita-gun, Kagawa 761-0795, Japan § JA-Nakanoshi, Miyoshino-cho, Nakano, Nagano 383-8588, Japan ABSTRACT: Here, we report the occurrence of the (2R,3S)-isomer of 2-amino-3,4-dihydroxybutanoic acid (D-ADHB) in the fruiting body of an edible mushroom, Hypsizygus marmoreus. This is an unusual example of the accumulation of a D-amino acid whose enantiomer is not a proteinogenic amino acid. We show that D-ADHB occurs specifically in the mushroom H. marmoreus. Other edible mushrooms examined, including Pholiota microspora, Pleurotus eryngii, Mycena chlorophos, Sparassis crispa, Grifola f rondosa, Pleurotus ostreatus, and Flammulina velutipes, do not contain detectable levels of D-ADHB. The concentration of DADHB in the fruiting body of H. marmoreus is relatively high (approximately 1.3 mg/g of fruiting body) and is comparable to the concentration of some of the most abundant free proteinogenic amino acids. Quantitative analysis of D-ADHB during fruiting body development demonstrated that the amino acid is synthesized during the fruiting body formation period. The absence of the putative precursors of D-ADHB, the (2S,3S)-isomer of ADHB and 2-oxo-tetronate, and the enzyme activities of D-ADHB racemase (2-epimerase) and transaminase suggested that D-ADHB is synthesized by a unique mechanism in this organism. Our data also suggested that the lack of or low expression of a D-ADHB degradation enzyme is a key determinant of D-ADHB accumulation in H. marmoreus. KEYWORDS: D-Amino acid, hydroxythreonine, Hypsizygus marmoreus, mushroom, fruiting body development

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INTRODUCTION Recent studies have demonstrated that many eukaryotes including mammals contain substantial amounts of free Damino acids which play important physiological functions. DSer, an endogenous D-amino acid produced by serine racemase (SR), is present at relatively high concentrations in the adult mammalian forebrain and acts as a coagonist of the N-methyl1−4 D-aspartate receptor. In the early postnatal cerebellum it serves as a ligand for the δ2-glutamate receptor and regulates long-term depression.5 D-Ser plays important roles in learning, memory, and behavior. In Arabidopsis thaliana, D-Ser controls pollen tube growth by activating glutamate receptor-like channels.6 D-Asp is another endogenous D-amino acid present in mammals;7 in the nervous system, it is involved in development and acts as a neurotransmitter.8,9 D-Asp is also found in the endocrine and exocrine systems, where it regulates hormone biosynthesis and secretion.10−13 Some aquatic crustaceans and bivalve mollusks accumulate D-Ala. It is synthesized by alanine racemase (AR) and functions as the major compatible osmolyte in these organisms.14,15 Here, we report the occurrence of an unusual D-amino acid, the (2R,3S)-isomer of 2-amino-3,4-dihydroxybutanoic acid (DADHB), in an edible mushroom, Hypsizygus marmoreus (the beech or bunashimeji mushroom). This is a rare example of the occurrence of the “noncanonical” D-amino acid. We also show for the first time that D-ADHB is synthesized in H. marmoreus during the fruiting body development period. Our data suggest that D-ADHB is synthesized by a unique mechanism, not by L© 2017 American Chemical Society

ADHB racemization or transamination of a corresponding keto acid. We discovered that unlike other mushrooms, H. marmoreus does not exhibit pyridoxal 5′-phosphate (PLP)dependent D-ADHB degradation activity, which may explain the accumulation of D-ADHB in H. marmoreus.

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RESULTS Occurrence of an Unusual D-Amino Acid in the H. marmoreus Fruiting Body. During the investigation of amino acids in edible mushrooms, we found that the cultivated fruiting body of H. marmoreus contained an unusual amino acid whose derivative was eluted at a retention time (RT) of 27 min in the amino acid analysis (Figure 1A). This amino acid was not detectable in the vegetative mycelium of H. marmoreus grown in a potato dextrose broth (Becton Dickinson, USA) (Figure 1B). Similarly, it was not detectable in the fruiting bodies or vegetative mycelia of the other mushrooms, Pholiota microspore, Pleurotus eryngii, Mycena chlorophos, Sparassis crispa, Grifola f rondosa, Pleurotus ostreatus, or Flammulina velutipes (Figure 1A and 1B). Subsequent studies demonstrated that this amino acid served as a substrate of D-amino acid oxidase (DAO) (Figure 2A) and D-amino acid aminotransferase (DAAT), indicating that the Received: Revised: Accepted: Published: 6131

April 25, 2017 July 6, 2017 July 7, 2017 July 7, 2017 DOI: 10.1021/acs.jafc.7b01893 J. Agric. Food Chem. 2017, 65, 6131−6139

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chromatogram of intracellular amino acid analyses of mushrooms. Amino acid composition in fruiting body (A) and vegetative mycelium (B). Mushrooms were cultivated as described in Materials and Methods. Mycelium and fruiting body were harvested and disrupted in TCA. Amino acids were derivatized with NAC/OPA and separated using a ODS column by HPLC. Lines indicated were (a) P. microspore, (b) P. eryngii, (c) H. marmoreus, (d) M. chlorophos, (e) S. crispa, (f) G. f rondosa, (g) P. ostreatus, and (h) F. velutipes.

Figure 2. Occurrence of D-amino acid in H. marmoreus. Amino acid extract of H. marmoreus fruiting body was incubated with DAO (A) and Dsd1p (B) and analyzed by HPLC. Broken lines indicate the samples after incubation of DAO or Dsd1p. Solid lines exhibit samples of negative control, in which boiled enzyme solution was used.

comparable to that of major amino acids including L-Glu, L-Gln, or L-Thr, as judged by the chromatogram of the amino acid analysis (Figures 1A and 2A). Cochromatographic analyses

amino acid has the D-configuration. This D-amino acid was present at a considerably high concentration in the fruiting body of H. marmoreus. Its intracellular concentration was 6132

DOI: 10.1021/acs.jafc.7b01893 J. Agric. Food Chem. 2017, 65, 6131−6139

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Figure 3. LC-ESI-MS analysis of AQC-derivative of the D-amino acid. Partially purified amino acid extract of H. marmoreus was incubated in the absence (a, b) or presence (c, d) of Dsd1p. Then amino acids were derivatized with AQC and separated with an ODS column (a, c). Eluates from the ODS column were collected every 1 min, and the fractions of RT 9−10 min (shaded) were subjected to an ESI-MS analysis (b, d). MS/MS spectrum of the ion of m/z 306.3 (marked with an arrow in b) was obtained (e). Putative structure of the AQC derivative of the target D-amino acid (m/z = 306) (left) and the daughter ion (m/z = 171) (right), corresponding to the AQC moiety of the derivative (f).

system (Figure 2A). These results suggest that the chemical structure of the D-amino acid is similar to that of L-Ser and/or L-Thr. We treated the D-amino acid with D-Ser dehydratase from Saccharomyces cerevisiae (Dsd1p). Dsd1p is a PLP- and Zn2+dependent enzyme that deaminates some D-amino acids that bear a Cβ-hydroxyl group, such as D-Ser, D-Thr, or D-allo-Thr, to produce a corresponding keto acid (pyruvate from D-Ser and 2ketobutyrate from D-Thr and D-allo-Thr) and ammonia.16,17 As shown in Figure 2B, the peak corresponding to the unusual Damino acid was no longer visible following Dsd1p treatment, strongly suggesting that the D-amino acid is an analog of D-Ser and/or D-Thr, and has a Cβ-hydroxyl group. Identification of the D-Amino Acid as (2R,3S)-2Amino-3,4-dihydroxybutanoic Acid. To obtain structural information on the H. marmoreus D-amino acid, we analyzed it by mass spectrometry (MS). The isolated D-amino acid was derivatized with 6-aminoquinolyl-carbamyl (AQC), separated using a C18 column (Figure 3A), and then subjected to MS with an electrospray ionization source (Figure 3B). As a negative control, the same experiment was conducted with the Dsd1p-treated sample (Figure 3C and 3D). Comparison of the MS spectra (Figure 3B and 3D) suggested that the m/z ratio of

with 19 proteinogenic amino acids of the D-form confirmed that the D-amino acid is not an enantiomer of a proteinogenic Lamino acid. D-Thr, D-ornithine, D-aminobutyric acid, and Dhomoserine (D-Hser) were also examined, but none of the Damino acids were coeluted with this D-amino acid (data not shown). These results indicate the occurrence of high levels of a novel D-amino acid in the fruiting body of H. marmoreus. Characterization of the D-Amino Acid in H. marmoreus. To the best of our knowledge, H. marmoreus is a rare example that contains a high concentration of an unusual Damino acid. Most of the D-amino acids studied are enantiomers of proteinogenic amino acids. To study this D-amino acid further, we sought to identify it. The D-amino acid of H. marmoreus was partially purified using cation and anion exchange chromatography. Then the major contaminants, L-Ser and L-Thr, were removed using LSer/L-Thr dehydratase. Using this procedure, the D-amino acid was separated from most of the other amino acids (data not shown). We noticed that the D-amino acid always fractionated with L-Ser and L-Thr in the purification procedure (which used ion exchange chromatography and thin layer chromatography). The NAC/OPA derivative of the D-amino acid was also eluted close to that of L-Ser on the reverse-phase chromatography 6133


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Figure 4. Identification of the D-amino acid as (2R,3S)-isomer of D-ADHB. (A) Scheme of enzymatic synthesis and degradation of (2R,3S)-ADHB. Briefly, L-Thr was hydroxylated by L-Thr hydroxylase to produce (2S,3S)-isomer of ADHB. Amino acid racemase converts the L-ADHB to (2R,3S)isomer of ADHB. Dsd1p dehydrates the D-ADHB to generate α-KHB. α-KHB was produced from D-Hser by DAAT. (B) Analysis of the keto-acid generated by the Dsd1p-treatment of H. marmoreus amino acid extract. Then keto-acid generated was derivatized with DMB and separated by a C18 column (line 2). α-KHB standard was generated from D-Hser with DAAT (line 4). Negative controls were performed in the absence of Dsd1p (line 1) and DAAT (line 3). (C) Comparison of RT of the D-amino acid in H. marmoreus (line 1) and enzymatically synthesized (2R,3S)-isomer of ADHB (line 2). D-ADHB (2R,3S) was prepared from L-ADHB (line 3, RT 15 min) by an amino acid racemase.

(Figure 4B, line 4). These results demonstrated that the Damino acid in H. marmoreus was D-ADHB. D-ADHB has 3S- and 3R-stereoisomers. To determine the stereochemistry at Cβ, we enzymatically synthesized the (2R,3S)-isomer of ADHB and analyzed its elution profile. Our HPLC analytical conditions could likely separate the 3R and 3S isomers of D-ADHB. A simple schematic of the enzymatic preparation of the (2R,3S)-isomer of ADHB is shown in Figure 4A. First, the (2S,3S)-isomer of L-ADHB (known as L-4-hydroxythreonine) was synthesized from L-Thr by using L-Thr hydroxylase, derived from Bordetella petrii DSM 12804 (BPE).18 Then it was epimerized by an amino acid racemase to produce the (2R,3S)-isomer of ADHB. As shown in Figure 4C, the elution profiles of the H. marmoreus D-amino acid derivative (line 1) and the enzymatically synthesized (2R,3S)-isomer of ADHB (line 2) were identical. Therefore, the D-amino acid in the H. marmoreus fruiting body is the (2R,3S)-isomer of ADHB. Biosynthesis of D-ADHB in H. marmoreus. To understand the origin of D-ADHB, we cultivated H. marmoreus and analyzed the time-dependent change of the D -ADHB

the ion of the AQC derivative was 306.3 or 262.3. It is known that most of the AQC-amino acid derivatives yield an identical daughter ion (m/z = 171) that corresponds to the AQC moiety of the derivative. MS/MS analyses demonstrated that the ion with an m/z ratio of 306.3 resulted in the daughter ion (m/z ratio of 171.2, Figure 3E), but the ion with the m/z ratio of 262.3 did not. The results indicate that the m/z ratio of the AQC-D-amino acid derivative was 306.3. We speculated that the unusual D-amino acid of H. marmoreus was D-2-amino-3,4-dihydroxybutanoic acid (hereafter referred to as D-ADHB, Figure 4A). D-ADHB likely exhibits chemical properties similar to those of Ser and Thr and results in the AQC derivative m/z ratio of 306. To verify this estimation, we analyzed the keto acid generated by Dsd1p treatment, wherein D-ADHB is expected to yield 2-keto-4hydroxybutanoic acid (α-KHB, Figure 4A). The keto acid was derivatized by 1,2-diamino-4,5-methylenedioxybenzene (DMB) and analyzed by HPLC. Under our analytical conditions, the derivative was eluted at an RT of 22.5 and 36.5 min (Figure 4C, line 2). The derivative of the α-KHB standard, which is generated from D-Hser by DAAT, exhibited an identical RT 6134


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Figure 5. Biosynthesis of D-ADHB and the concentration change during the development. Concentration of D-ADHB in 41 days mycelium and primordium (a), 52 days mycelium (b) and immature fruiting body (c), 60 days mycelium (d), mature fruiting body (pileus) (e), and fruiting body (stipe) (f) were determined by HPLC using NAC/OPA derivatization. Closed arrow shown in A indicates the derivative of D-ADHB. Open arrow shows the RT of the derivative of L-ADHB. In B, the concentration of D-ADHB at 60 days fruiting body (stalk) (approximately 10 μmol/g fruiting body as judged by the peak area of HPLC analysis), is represented as 100%.

concentration during the fruiting body development. Briefly, H. marmoreus mycelium was inoculated into a cultivation medium and incubated at 23 °C for 26 days. Then fruiting body formation was induced by a low-temperature shift (from 23 to 15 °C) and lighting. In our experiment, the primordium was observed at 41 days. The immature fruiting body became visible at 52 days. Samples were collected from the upper surface of the medium at 41, 52, and 60 days, and the intracellular concentrations of D-ADHB were analyzed. As shown in Figure 5, in the 41- and 52-day mycelia and primordia, trace amounts of D-ADHB were detected (line A and line B, and Figure 5B). At 52 days, the immature fruiting body contained a considerable amount of D-ADHB (line C). The highest intracellular concentration was detected in the stipe of the mature fruiting body at 69 days (line F). The level of D-ADHB was comparable to that of the most abundant amino acid, LGlu, and it was approximately 10 μmol/g (1.3 mg/g fruiting body), as approximated from the peak area of the chromatogram. No free D-ADHB was detected in the medium used in this experiment (Figure 5B). These results clearly demonstrate that D-ADHB is an endogenous D-amino acid and is synthesized during H. marmoreus fruiting body development. H. marmoreus Cell-Free Extract Lacks the D-ADHB Degrading Enzyme. The mechanism by which H. marmoreus synthesizes D-ADHB is interesting. In most cases, PLPindependent and/or -dependent amino acid racemases are responsible for the production of a D-amino acid,19 with the corresponding L-amino acid as the direct precursor. However, the amino acid analyses of H. marmoreus (41-day mycelium and primordium, 52-day mycelium and fruiting body, and 69-day

mycelium and fruiting body) demonstrated that during development it contains little of the (2S,3S)-isomer of LADHB (Figure 5A). This implies that D-ADHB is not produced directly from L-ADHB. We prepared a cell-free extract of the H. marmoreus fruiting body and incubated it with D-ADHB in the presence or absence of PLP. In all cases, the level of D-ADHB in the reaction mixture remained unchanged following overnight incubation with the cell-free extract. This indicates the absence of or low-level expression of enzymes catalyzing racemization, decarboxylation, and deamination of D-ADHB in the fruiting body of H. marmoreus. DAO and/or DSD are plausible degradation enzymes for DADHB. In mammals, DAO acts as a primary enzyme that degrades endogenous D-Ser (DSD is absent in mammals).20 In other eukaryotes, DSD likely degrades D-Ser and probably plays a significant role in the low-level maintenance of cellular DSer.21 We performed similar experiments using the cell-free extracts of the P. microspore, P. ostreatus, and F. velutipes fruiting bodies. All of these cell-free extracts exhibited D-ADHB degradation activity. It should be noted that in the P. microspore and P. ostreatus cell-free extracts, D-ADHB degradation activity was PLP dependent. A database search indicated that many mushrooms possess the Dsd1p homologue (some examples are shown in Figure 6). This may indicate that the D-ADHB degradation activity observed in the P. microspore, P. ostreatus, and F. velutipes varieties originated from Dsd1p homologues. The lack of or low-level expression of D-ADHB-degrading enzyme(s) in the fruiting body is a possible reason for the accumulation of D-ADHB in H. marmoreus. 6135


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Figure 6. Sequence alignment of Dsd1p homologues in mushrooms. Amino acid sequences of the Dsd1p homologues in H. marmoreus, G. f rondosa, P. ostreatus, and F. velutipes. Similar residues are boxed in gray, and identical residues are boxed in black. Active site residues are marked with circles. Amino acid sequences were aligned using ClustalW (http://www.genome.jp/tools/clustalw/), and residues were shaded using the BoxShade (http://embnet.vital-it.ch/software/BOX_form.html) programs.

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DISCUSSION The present study demonstrated the specific accumulation of an unusual D-amino acid, the (2R,3S)-isomer of ADHB, in an edible mushroom, H. marmoreus. After its identification, we learned that Ogawa et al. reported the detection of D-ADHB in Lyophyllum ulmarium, a close relative of H. marmoreus.22 Other than these two organisms, there are no reports of the occurrence of D-ADHB. As described, most of the D-amino acids studied in eukaryotes thus far are enantiomers of the proteinogenic amino acids. H. marmoreus and L. ulmarium are unique examples of the occurrence of noncanonical D-amino acids. In addition, to the best of our knowledge, no other organism has been reported to accumulate L-ADHB. Our data demonstrate that D-ADHB is synthesized during fruiting body development. This suggests that D-ADHB may have biological importance during H. marmoreus development, but this remains to be elucidated. It is known that some Damino acids are components of various bioactive peptides.23,24 In addition, many D-amino acids are known to inhibit bacterial

growth by hampering cell wall synthesis, protein synthesis, and/ or metabolic processes.25−27 It is known that the (2S,3S)isomer of ADHB is a toxic antimetabolite, probably because its structure is similar to those of L-Ser, L-Thr, L-Hser, and/or L-4hydroxythreonine-4-phosphate, an intermediate of vitamin B6 biosynthesis.28 We found that D-ADHB is secreted into the cultivation medium during development (data not shown). This led us to speculate that D-ADHB is an endogenous antibacterial toxin, and probably, the intra- and extracellular DADHB prevents bacterial growth. Ogawa et al. discovered the presence of O4-ethoxylated D-ADHB in L. ulmarium.29 In some plants, N-malonylated-D-amino acids such as D-malonyl-D-Trp and D-malonyl-D-Ile have been detected, and their derivatives are suggested to have important biological functions.24 It is possible that D-ADHB serves as a precursor for O4-ethoxylated D-ADHB, although it is currently unknown whether H. marmoreus accumulates O4-ethoxyl-D-ADHB. It is also possible that ethoxyl-D-ADHB, not D-ADHB, is bioactive. Kawagishi et al. reported that D-Ile accelerates the growth of the mycelia of 6136



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Tricholoma matsutake (Matsutake mushrrom) at a very low concentration.30 Structural analogy of D-ADHB to D-Ile may suggest the plausible role of D-ADHB and/or its derivative as an endogenous growth activator in H. marmoreus. Our data indicate that D-ADHB is an endogenous D-amino acid, but the enzyme(s) responsible for its biosynthesis is currently unknown. Elucidation of the D-ADHB metabolic enzymes will shed light on the biological significances of this Damino acid. The simplest and most direct way of producing DADHB is by the racemization (2-epimerization) of the (2S,3S)isomer of ADHB. Indeed, most free D-amino acids are synthesized by amino acid racemases. Interestingly, the LADHB was not detected in H. marmoreus under our experimental conditions. Homologues of AR and mammalian SR that are responsible for D-Ala and D-Ser/D-Asp production, respectively, in many eukaryotic organisms are not encoded in the genome of H. marmoreus. In addition, ADHB racemase activity was not detected under our analytical conditions. Although we cannot deny the presence of ADHB racemase, our data suggest that D-ADHB is not synthesized through its action. It is also conceivable that D-ADHB is produced by DAAT. Although we examined the occurrence of the corresponding αketo acid, oxo-tetronate, no detectable amount of the keto acid was detected during H. marmoreus development (data not shown). These data suggest that, unlike other D-amino acids, DADHB is not synthesized by the actions of racemase and transaminase in H. marmoreus. In mammals, D-Ser and D-Asp degradation enzymes (DAO and D-Asp oxidase, respectively) play an important role in the regulation of D-Ser and D-Asp concentrations.20,31,32 Our in vitro analyses failed to detect D-ADHB degradation activity in the fruiting body of H. marmoreus. In contrast, D-ADHB degradation activity was detected in other mushrooms including P. microspore, P. ostreatus, and F. velutipes under the same analytical conditions. This indicates the lack of or lowlevel expression of a D-ADHB degradation enzyme in the H. marmoreus fruiting body. However, the genome of H. marmoreus encodes four putative D-ADHB degradation enzymes, a Dsd1p homologue (GenBank ID KYQ45893.1, hereafter referred to as HmDsd) and three putative DAOs (GenBank ID KYQ35714.1, KYQ34666.1, and KYQ34720.1). Judging by the primary structure, these putative enzymes are likely capable of degrading D-ADHB. Because Dsd is much more active toward D-Ser and D-Thr than DAO, we expect that of the four putative enzymes HmDsd will play a major role in DADHB degradation. Figure 6 demonstrates the sequence alignment of the Dsd1p, HmDsd, and Dsd1p homologues in G. f rondosa, P. ostreatus, and F. velutipes. HmDsd has a long Nterminal extension and exhibits 25%, 63%, 59%, and 60% sequence identity with Dsd1p and the homologues of G. f rondosa, P. ostreatus, and F. velutipes, respectively. The putative active site residues including PLP-binding Lys (K109 in HmDsd), Zn2+-binding residues (H438 and C440), and some other residues in the active site (H253 and Y258) are highly conserved in these proteins. This suggests that the HmDsd may catalyze D-ADHB dehydration. The temporal and spatial expression and the substrate specificities of HmDsd and the DAO homologues are currently unknown. Characterization of the putative enzymes will shed light on the metabolic pathway and the physiological roles of D-ADHB in H. marmoreus.

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MATERIALS AND METHODS

Chemicals. Amino acids, N-acetyl-L-cysteine, o-phthalaldehyde were obtained from Wako (Japan). 1,2-Diamino-4,5-methylenedioxybenzene was from Dojindo (Japan). Synthetic oligonucleotide was purchased from Operon-Eurofins (Japan). Cultivation of Mushrooms. In this study, we used the following mushroom strains; Hypsizygus marmoreus NN-12, Flammulina velutipes SR-12, Pleurotus eryngii K1W, Pleurotus ostreatus HR (JA-Nakanoshi, Japan), Grifola f rondosa Mori-51, Pholiota microspora Mori-N11 (Mori Sangyo, Japan), Sparassis crispa KSC-H7 (Katsuragi Sangyo, Japan), and Mycena chlorophos NBRC31858 (National Institute of Technology and Evaluation, Japan). The above mushrooms, except for M. chlorophos, were cultivated in a conventional manner using the sawdust medium supplemented with rice bran. M. chlorophos was cultivated according to the method previously reported.33 Enzyme Preparation. D-Serine dehydratase (DSD) from Saccharomyces cerevisiae (Dsd1p) was prepared as described in ref 34. DAmino acid oxidase (DAO) from Shizosacchromyces pombe was purified according to the method described in ref 35. Escherichia coli L-threonine dehydratase (IlvA, N-terminal His-tag) was overexpressed in E. coli AG1 with the pCA24N-ilvA plasmid (ASKA (−) clone JW3745-AM,36 NBRP (NIG, Japan): E. coli). A C-terminal His6tagged mutated alanine racemase (AR) of Geobacillus stearothermophilus (I222T/Y354W mutant)37 and a C-terminal His6-tagged Damino acid transaminase (DAAT) of Bacillus stearothermophilus38 were overexpressed in E. coli KRX by using pET22b vector. N-Terminal His6-tagged L-threonine hydroxylase from Bordetella petrii DSM 12804 (BPE)18 was expressed in E. coli KRX with pET15b. The L-threonine hydroxylase gene was synthesized using GeneArt Strings (Invitrogen), in which codon usage was optimized for expression in E. coli. All E. coli strains were cultivated in LB medium supplemented with ampicillin (100 μg/mL) or chloramphenicol (30 μg/mL). Expression of all of the His-tagged enzymes were induced by adding 0.1% L-rhamnose (E. coli KRX) or 0.5 mM isopropyl-β-D-thiogalactopyranoside (E. coli AG1) at mid log phase. After the induction, E. coli cells were cultivated at 22◦C for another 16 h. Cells pellet was disrupted by sonication, and the Histagged enzymes were purified homogeneity by a Ni-affinity chromatography. Amino Acid Analysis. Mycelium and/or fruiting body of mushrooms was disrupted in 10 vol. (10 μL/1 mg wet weight) of 5% trichrolo acetic acid (TCA) by using a pestle and mortar or bead beating (Bead-Smash 12, Wakenyaku, Japan). TCA was removed from the samples by three-times extraction with water-saturated diethyl ether. They were dried by a centrifugal evaporator and dissolved with distilled water (amino acid extract). Amino acids were derivatized by N-acetyl-L-cysteine (NAC) and o-phthalaldehyde (OPA) and separated by HPLC as previously described.39,40 The (2R,3S)-isomer of 2amino-3,4-dihydroxybutanoic acid (D-ADHB) was enzymatically synthesized from L-Thr. L-Thr was reacted with BPE as described in ref 18 and incubated at 37◦C for 16 h. The reaction was terminated by boiling. The reaction mixture contained L-Thr and (2S,3S)-isomer of ADHB (L-ADHB). Then the reaction mixture (100 μL) was incubated with 30 μg of the I222T/Y354W mutant of AR.37 The final reaction mixture contained L-Thr, D-allo-Thr, L-ADHB, and D-ADHB. The retention time of the D-allo-Thr derivative was confirmed by cochromatography with D-allo-Thr standard. The concentrations of D-ADHB were estimated by using peak areas of a known concentration of L-Thr derivative. Enzyme Treatment of Amino Acid Extract. Amino acid extract was incubated with DAO, DAAT, DSD, or IlvA (∼5 μg) in a buffer consisting of 50 mM potassium phosphate buffer pH 7.8 (50 μL) at 30 °C for 16 h. Boiled enzyme was used as a negative control. Amino acid extract was also incubated with cell-free extract of mushrooms. The cell-free extract was prepared by disrupting the edible portion of mushrooms (mostly fruiting body) in a buffer of 50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 10% glycerol, and 1× protease inhibitor (nachalai tesque, Japan) with the HBB250SR homogenizer (Hamilton Beach, Germany) and the following centrifugation (20 000g, 4◦C, 20 min). Reaction was terminated by adding the same amount of 10% 6137


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TCA. After removal of the TCA by ether extraction, amino acids concentrations were determined by HPLC with NAC and OPA. LC-ESI-MS Analysis. 6-Aminoquinoline N-succinimidyl ester (AQC) was synthesized as described previously.41 Partially purified amino acid sample was incubated with or without Dsd1p (15 μg) in a 100 mM potassium phosphate buffer (100 μL, pH 7.8) for 16 h at 30 °C. Reaction was terminated by boiling for 5 min. Then a 45 μL amount of the portion was mixed into 35 μL of 0.4 M borate buffer pH 9.5, and 20 μL of AQC solution (3 mg/mL in dry acetonitrile) was added. It was incubated at 55 °C for 10 min. After centrifugation for 15 min at 20 000g, a 100 μL amount of Buffer A (0.14 M ammonium acetate, pH 5.05) was added to the solution. The AQC derivatives were separated by HPLC with a Kinetex XB C18 column (4.6 × 250 mm, 5 μm) (Phenomenex, USA) and two buffers, Buffer A and Buffer B (acetonitrile/water = 60/40, v/v). Time programs (linear gradient) used for separation of the AQC derivatives were as follows: time (min)/%B, 0 min/12.5%, 20 min/12.5%, 25 min/19%, 40 min/43%. Flow rate was 0.8 mL/min, and the column was maintained at 37 °C. The excitation and emission wavelengths were 250 and 395 nm, respectively. The eluate was collected every 1 min and analyzed by direct infusion ESI-MS in a positive mode with an Esquire 3000 ion trap system (Bruker Daltonics, USA). Keto Acid Analysis. The partially purified amino acid extract of H. marmoreus fruiting body was reacted with DSD (15 μg) in a buffer of 50 mM potassium phosphate (100 μL, pH 7.8) at 37 °C for 16 h. The 2-keto-3,4-dihydroxybutanoic acid standard was obtained by incubation of D-homoserine (D-Hser) with DAAT. The reaction mixture (100 μL) was 100 mM potassium phosphate pH 8.0, 0.1 mM D-Hser, 1 mM pyruvate, and 15 μg DAAT and incubated at 37 °C for 16 h. The keto acid formed was reacted with 1,2-diamino-4,5-methylenedioxybenzene (DMB), and the DMB derivative was analyzed by HPLC as described previously.35

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +81 87 891 3112; Fax: +81 87 891 3021. E-mail: [email protected]. *Tel.: +81 52 789 4132; Fax: +81 52 789 4120. E-mail: [email protected]. ORCID

Tohru Yoshimura: 0000-0003-4871-1082 Author Contributions

T.I., Y.A., and T.Y. designed research; T.I., Y.Z., I.Y., Y.H., K.Y. performed research; T.I., Y.Z., I.Y., Y.H., K.Y., K. S., A.W., H.H., Y.A., and T.Y. analyzed data; T.I., Y.A., and T.Y. wrote the paper. Notes

This study involved neither human participants nor animals. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by JSPS KAKENHI Grant nos. 16H04908 (to T.Y.) and 16K18686 (to T.I.) and by funding from the Hokuto Foundation for Bioscience (to T.I.).

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ABBREVIATIONS ADHB, 2-amino-3,4-dihydroxybutanoic acid; PLP, pyridoxal 5′phosphate; AQC, 6-aminoquinolyl-carbamyl; DAO, D-amino acid oxidase; Dsd, D-serine dehydratase

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REFERENCES

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