JB
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Other Versions of this Article:
JB.01184-07v1
190/4/1359    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohnishi, M.
Right arrow Articles by Kasai, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohnishi, M.
Right arrow Articles by Kasai, S.

 Previous Article  |  Next Article 

Journal of Bacteriology, February 2008, p. 1359-1365, Vol. 190, No. 4
0021-9193/08/$08.00+0     doi:10.1128/JB.01184-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of Serine Racemase from a Hyperthermophilic Archaeon, Pyrobaculum islandicum{triangledown} ,{dagger}

Masato Ohnishi,1 Makoto Saito,1 Sadao Wakabayashi,2 Morio Ishizuka,3 Katsushi Nishimura,4 Yoko Nagata,1* and Sabu Kasai5*

Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-8-14 Kanda-Surugadai, Chiyoda-Ku, Tokyo 101-8308, Japan,1 Department of Life Sciences, Graduate School of Life Science, University of Hyogo, Kamigohri, Hyogo, Japan,2 Department of Applied Chemistry, Faculty of Science and Engineering, Chuo University, Bunkyo-Ku, Tokyo, Japan,3 Department of Applied Chemistry, Junior College, Nihon University, Funabashi, Chiba, Japan,4 Department of Applied Chemistry and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, Sugimoto, Sumiyoshi, Osaka 558-8585, Japan5

Received 26 July 2007/ Accepted 12 October 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pyrobaculum islandicum is an anaerobic hyperthermophilic archaeon that is most active at 100°C. A pyridoxal 5'-phosphate-dependent serine racemase called Srr was purified from the organism. The corresponding srr gene was cloned, and recombinant Srr was purified from Escherichia coli. It showed the highest racemase activity toward L-serine, followed by L-threonine, D-serine, and D-threonine. Like rodent and plant serine racemases, Srr is bifunctional, showing high L-serine/L-threonine dehydratase activity. The sequence of Srr is 87% similar to that of Pyrobaculum aerophilum IlvA (a putative threonine dehydratase) but less than 32% similar to any other serine racemases and threonine dehydratases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses revealed that Srr is a homotrimer of a 44,000-molecular-weight subunit. Both racemase and dehydratase activities were highest at 95°C, while racemization and dehydration were maximum at pH 8.2 and 7.8, respectively. Unlike other, related Ilv enzymes, Srr showed no allosteric properties: neither of these enzymatic activities was affected by either L-amino acids (isoleucine and valine) or most of the metal ions. Only Fe2+ and Cu2+ caused 20 to 30% inhibition and 30 to 40% stimulation of both enzyme activities, respectively. ATP inhibited racemase activity by 10 to 20%. The Km and Vmax values of the racemase activity of Srr for L-serine were 185 mM and 20.1 µmol/min/mg, respectively, while the corresponding values of the dehydratase activity of L-serine were 2.2 mM and 80.4 µmol/min/mg, respectively.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pyrobaculum islandicum is an anaerobic rod-shaped archaeon that grows optimally at 100°C; it was isolated from samples taken at a geothermal power plant in Iceland (11). P. islandicum grows facultatively chemolithoautotrophically through the reduction of elemental sulfur by hydrogen or organotrophically by using complex organic compounds as hydrogen donors and producing H2S (11). Although the metabolism of hyperthermophilic archaea is of interest, since they may be among the most primitive and ancestral organisms on earth, not many studies have been performed on them. Thus, knowledge of the amino acid metabolism of archaeal cells is rather limited.

D-Enantiomers of amino acids have not been considered a possible constituent of archaeal cells because they possess no peptidoglycan layer (15). However, in P. islandicum cells, we have found free D-serine at quite high concentrations and have detected D-amino acid dehydrogenase activity that catalyzes the catabolism of free neutral D-amino acids (20). This finding prompted us to search the organism for a serine racemase that produces D-serine from its L-enantiomer. Although threonine dehydratase/serine racemase genes have been cloned from a wide range of archaea, none of their gene products have been isolated from the original archaeal cells or expressed in Escherichia coli to obtain details about their characteristics. In the present study, we purified the enzyme Srr and cloned its gene, srr. Recombinant Srr is a pyridoxal 5'-phosphate (PLP)-dependent enzyme exhibiting both L-serine dehydratase and racemase activities. As far as we know, this is the first detailed report on the enzymatic properties of a serine racemase/dehydratase from archaea.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. P. islandicum DSM 4184 was obtained from the Japan Collection of Microorganisms (JCM 9189; Wako, Saitama, Japan) and was grown anaerobically in a 5-liter glass bottle (with a slightly loosened top to release the H2S gas produced in the culture) for 24 h at 95°C in an electric oven (NDO-500; Tokyo Rika Kikai, Tokyo, Japan). The culture medium was modified slightly from the one described by Huber et al. (11) in order to reduce the amount of black FeS sediment formed during the culturing: it contained 0.05% (wt/vol) peptone (Oxoid, Hampshire, United Kingdom), 0.02% yeast extract (Oxoid), 0.13% (NH4)2SO4, 0.025% MgSO4·7H2O, 0.025% KH2PO4, 0.2% Na2S2O3·5H2O, 0.05% L-cystine, basal minerals (11), and resazurin. The pH of the culture medium was adjusted to 7.0 with H2SO4 and NaOH. Cells were harvested by continuous centrifugation (H-600N; Kokusan, Tokyo, Japan) at 16,000 x g for 10 min and washed once with 50 mM sodium phosphate buffer (pH 7.0) before being stored at –80°C.

Recombinant E. coli Rosetta 2 (Merck, Darmstadt, Germany) cells harboring pET-21dPisrr were cultured on LB medium containing 100 µg/ml ampicillin and 25 µg/ml chloramphenicol in Sakaguchi flasks at 37°C for 8 h with continuous shaking. The cells were collected by centrifugation at 10,000 x g for 10 min and washed once with 50 mM sodium phosphate buffer (pH 7.0) before being stored at –80°C.

Purification of enzyme from recombinant E. coli. Recombinant E. coli cells (wet weight, 4.2 g) were suspended in 50 mM sodium phosphate buffer (pH 7.2) containing 10 µM PLP and 1 mM phenylmethanesulfonyl fluoride and disrupted by sonication with a Sonifier 450 (Branson, Connecticut) for 15 min (15 1-min rounds of sonication at 30-s intervals) at 20 kHz. Cell debris and intact cells were spun off by centrifugation at 10,000 x g for 20 min, and the resultant pellet was suspended in 20 ml of 50 mM sodium phosphate buffer (pH 7.2) containing 2 M L-arginine at 50°C. After 5 h, the suspension was centrifuged at 10,000 x g for 20 min. The supernatant was dialyzed against 1,000 ml of 20 mM Tris-HCl buffer (pH 8.5) for 6 h with three buffer changes and loaded on a Mono Q column (5 mm [inside diameter] by 50 mm) equilibrated with the same buffer. The adsorbed portion was eluted with a linear gradient of 0 to 200 mM NaCl in the same buffer.

Racemase activity. The assay mixture for racemase contained 0.5 to 50 µg of an enzyme sample, 400 mM L-serine, and 10 µM PLP in 200 µl of 100 mM Tris-HCl buffer (pH 8.2) unless otherwise indicated. After incubation at 95°C for 30 min, 100 µM NH2OH and 5% trichloroacetic acid were added to stop the reaction and to remove proteins. The supernatant yielded by centrifugation at 4,000 x g for 10 min was analyzed for D- and L-serine using high-performance liquid chromatography (21) after its amino acids were modified with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent [17]; Sigma, St. Louis, MO), and the 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide serine was isolated on a thin-layer chromatography plate (Merck).

Dehydratase activity. The assay mixture for dehydratase contained 0.5 to 10 µg of enzyme sample, 10 mM L-serine, 10 µM PLP in 200 µl of 100 mM Tris-HCl buffer (pH 7.8) unless otherwise indicated. After being incubated at 95°C for 10 min, the assay mixture was rapidly cooled to 30°C by the addition of 800 µl of 100 mM cold acetate buffer (pH 5.0) containing 0.5 U of L-lactate dehydrogenase (Sigma) and 2 mM NADH (Sigma). The initial velocity of decrease in absorbance at 340 nm was measured with an UltroSpec spectrophotometer (Amersham BioSciences). The amount of pyruvate produced from L-serine was calculated based on a standard curve for pyruvate.

Protein concentrations were determined by the Bradford method (3) using bovine serum albumin as a standard.

SDS-PAGE. Electrophoresis was carried out with a 12.5% polyacrylamide gel, according to Laemmli's method (16). A low-molecular-weight calibration kit for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Amersham BioSciences) was used for molecular marker proteins. The proteins were stained with Coomassie brilliant blue.

Molecular weight estimation. A Shodex KW-2003 gel filtration column was calibrated using molecular weight marker proteins (Oriental Yeast, Tokyo, Japan), such as yeast glutamate dehydrogenase (290,000 [290K]), porcine heart lactate dehydrogenase (142K), yeast enolase (67K), yeast myokinase (32K), and cytochrome c (12.4K), in the presence of 50 mM sodium phosphate buffer (pH 7.2) containing 200 mM NaCl.

The molecular weight of native serine racemase was also determined by low-angle laser light-scattering and differential-refraction measurements, combined with gel chromatography as described previously (9, 31), using a TSKgel SuperSW3000 column (4.6 by 300 mm; Tosoh). The ratio of the output of the light-scattering photometer to that of the refractometer was plotted against the molecular weight.

N-terminal protein sequencing. The proteins separated by SDS-PAGE were transferred to a polyvinylidene fluoride membrane (Sequi-Blot; Bio-Rad, Munich, Germany) using a semidry system and stained with Coomassie brilliant blue. The bands were cut out, and the N-terminal amino acid sequence of the protein was analyzed with an automated Edman degradation protein sequencer (PPSQ-1; Shimadzu, Kyoto, Japan).

Cloning of the serine racemase gene from P. islandicum. The conditions for PCR and DNA sequencing were reported previously (13). Sequencing of the gene for serine racemase (srr) of P. islandicum with both flanking regions is summarized in Fig. 1A. A partial sequence of srr (Seq1; 464 bp) was amplified by PCR with P. islandicum genomic DNA, isolated using a GenTLE yeast kit (Takara Shuzo, Kyoto, Japan), as a template. In the PCR, a pair of degenerate primers was used, i.e., 5'-AAYCAYGCNCARGGNGTNGC-3' and 5'-ATNGCDATNCCRTCNGCDAT-3'. The pair was designed based on the two conserved amino acid sequences, NHAQGVA and IADGIAI, respectively, from among the various archaeal proteins encoded by threonine dehydratase (ilvA) genes. An upstream sequence (Seq2; 373 bp) was amplified using a nondegenerate primer, 5'-ACTCCATGTAATTGTGCGGC-3', and a degenerate primer, 5'-GCNATGCGNCTNAAYTTYAC-3', which was designed based on the conserved amino acid sequence, AMRLNFT, in the gene product of PAE2315, present just upstream of ilvA in P. aerophilum. A downstream sequence (Seq3; 380 bp) was amplified using a nondegenerate primer, 5'-TCACTTAAAGAGGGGAAGCC-3', and a degenerate primer, 5'-GTNCCNGGYTTRTCNGGNAC-3', which was designed based on the conserved amino acid sequence, VPDKPGT, in IlvA proteins. A Seq4 sequence was determined by SUGDAT (Sequencing Using Genomic DNA as a Template) (12, 14) using the primer 5'-CGTATCATAAAACTAGTCGG-3'. All PCR products were sequenced on both strands without cloning in order to prevent reading errors.


Figure 1
View larger version (23K):
[in this window]
[in a new window]

  		FIG. 1. (A) Outline of sequencing of P. islandicum srr with both flanking regions. Three sequences, Seq1, Seq2, and Seq3, indicated by open bars, were amplified by PCR and sequenced as described in Materials and Methods. Seq4, indicated by an open arrow, was sequenced in this direction by SUGDAT. (B) Construction of a pET-21d derivative containing the P. islandicum srr sequence. P. islandicum srr with both flanking regions was amplified by PCR using a high-fidelity DNA polymerase and digested by two restriction enzymes, BspHI and SacI, while pET-21d was digested by NcoI and SacI. The respective digests were purified by electrophoresis and ligated with each other.

 
A scheme for constructing a pET-21d plasmid harboring the srr sequence is shown in Fig. 1B. P. islandicum srr with the 6-bp upstream sequence, along with the 18-bp downstream sequence (1,233 bp), was amplified by PCR using 5'-AAGTTCATGATATTACTAGAAGAGGCAC-3' and 5'-CCAAGGAGCTCTACAACATTAAGCC-3' as primers, the genomic DNA of P. islandicum as a template, and a high-fidelity DNA polymerase, GeneAmp (Applied Biosystems, California), according to the instructions provided. The PCR product was digested with two restriction enzymes, BspHI (New England Biolabs, Suffolk, United Kingdom) and SacI (New England Biolabs). After purification by gel electrophoresis, the resulting fragment was ligated into the vector pET-21d (Novagen, Madison, WI), which had been digested beforehand by the two restriction enzymes, NcoI (New England Biolabs) and SacI. After purification by gel electrophoresis, the completed plasmid, designated pET-21dPisrr, was transferred into competent cells of E. coli Rosseta 2 and amplified. All PCR products cloned into the plasmids were sequenced on both strands to confirm that no mutations had been introduced during PCR amplification.

Nucleotide sequence accession number. The nucleotide sequence of P. islandicum serine racemase has been deposited in the GenBank/DDBJ/EMBL database under accession number AB244101.

Protein structure accession number. The amino acid sequence of P. islandicum serine racemase can be accessed through the NCBI Protein Database under accession number BAE54303.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Purification of serine racemase from P. islandicum. The purification method and results are summarized in the supplemental material. The low specific activity of the final preparation (1.91 µmol/min/mg) is ascribed to the fact that it contained another protein (Fig. 2) and the concentration of L-serine used in the assay was lower than the Km. Throughout the purification, L-serine dehydratase activity accompanied and increased in tandem with the serine racemase activity. The final preparation showed two protein bands, as analyzed by SDS-PAGE (Fig. 2). The N-terminal amino acid sequence analysis of the lower band (with a larger Rf value) revealed MILLEEALSVIRE, which is highly similar to the N-terminal sequence, MILLEEAASVIRE, deduced from the nucleotide sequence of the P. aerofilum ilvA gene assigned to a threonine dehydratase.


Figure 2
View larger version (74K):
[in this window]
[in a new window]

  		FIG. 2. SDS-PAGE of P. islandicum serine racemase Srr. Lanes: 1, the final preparation from P. islandicum cells; 2, cell extract (20 µg) of E. coli Rosetta 2 cells harboring pET-21dPisrr; 3, cell debris and unbroken cells of the above-mentioned E. coli Rosetta 2 cells; 4, L-arginine-solubilized fraction (2 µg) from the above-mentioned E. coli cell debris; 5, L-arginine-insoluble fraction (5 µg); 6, Mono Q-column-eluted solution (2 µg) of L-arginine-solubilized fraction; 7, molecular marker proteins.

 
The activities of the racemase and dehydratase were completely inhibited by both 1 mM hydroxylamine and 1 mM sodium borohydride, inhibitors of a PLP-dependent enzyme. Neither enzyme's activity was affected by other SH reagents, such as p-chloromercuribenzoate (5 mM), iodoacetamide (10 mM), and iodoacetate (10 mM).

Sequencing of the P. islandicum serine racemase gene. To identify a gene for the serine racemase of P. islandicum, we sequenced an orthologue of ilvA and named the gene srr. DNA sequence analysis of srr showed the predicted start codon, ATG, and an open reading frame composed of 1,289 bases. The amino acid sequence deduced from the nucleotide sequence of this frame is given in Fig. 3. The N-terminal 13-amino-acid sequence, MILLEEALSVIRE, coincided with that of the serine racemase purified from P. islandicum. The primary structure of this sequence is compared to the structures of other serine racemases and related enzymes in Table 1. The similarities are relatively low, except with P. aerophilum IlvA. The protein has no cysteine residue, which explains why none of the SH reagents affected the enzymatic activity.


Figure 3
View larger version (28K):
[in this window]
[in a new window]

  		FIG. 3. Deduced amino acid sequence of the P. islandicum srr gene-encoded protein. The underlining indicates well-conserved sequence motifs among many serine/threonine dehydratases. *, Conserved lysine residue that participates in formation of a Schiff base.

 

View this table:
[in this window]
[in a new window]

  		TABLE 1. Amino acid sequence similarity of P. islandicum Srr with other racemases and dehydratases

 
Purification of serine racemase from recombinant E. coli cells. The srr gene was expressed in E. coli Rosetta 2 cells. The enzyme activity was detected, not in the cell extract, but in the fraction solubilized from the centrifuged pellet by L-arginine treatment. The recombinant enzyme, Srr, was purified to homogeneity by the following column chromatography with Mono Q, as determined by SDS-PAGE (Fig. 2). The specific activities of Srr for racemization and dehydration are shown in Table 2. The molecular weight of Srr was estimated to be 44K by SDS-PAGE analysis (Fig. 2), showing a good coincidence with the value 44,051, calculated based on the primary structure deduced from srr (Fig. 3). The molecular weight of native Srr obtained with gel filtration through the Shodex KW-2003 column was 137K. The molecular weight was also estimated using the light-scattering-refraction method, resulting in a similar value, 143K ± 22K (data not shown). The possibility is low that the sample was an equilibrium mixture of dimers and tetramers because the retention time of the sample in the gel column did not change with its concentration (0.1 to 0.2 mg/ml).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Purification of P. islandicum Srr from recombinant E. coli cells

 
The absorption spectrum of Srr (Fig. 4), with a broad peak around 410 nm caused by a Schiff base, clearly reveals that the protein is a PLP enzyme. This was demonstrated by the complete disappearance of the peak upon the addition of hydroxylamine.


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 4. Absorption spectra of P. islandicum Srr. Srr (0.5 mg) was dissolved in 1 ml of 100 mM Tris-HCl buffer (pH 8.0) containing 10 µM PLP and measured with an Ultrospec4000 (Amersham Biosciences) spectrophotometer before (solid line) and after (dashed line) addition of 1 mM hydroxylamine.

 
The optimum pH values for Srr were 8.2 for racemase and 7.8 for dehydratase activities (Fig. 5A). Both of the enzyme activities increased with temperature elevation and showed the highest levels at 95°C (Fig. 5B). These values coincided well with those obtained with the protein purified from the P. islandicum cells. The stability of Srr dehydratase activity was examined. The remaining activities after an 8-h incubation at 50°C, 75°C, and 95°C in a 100 mM Tris-HCl buffer (pH 7.8) containing 10 µM PLP were 95.2%, 67.1%, and 29.8%, respectively (data not shown).


Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 5. (A) pH dependency of P. islandicum Srr activity. The buffers used were 100 mM MOPS (morpholinepropanesulfonic acid)-NaOH (circles) and 100 mM Tris-HCl buffer (squares) containing 10 µM PLP and 400 mM L-serine or 10 mM L-serine, respectively, for the racemase (open symbols) or dehydratase (solid symbols) assay. Aliquots of 0.6 µg of Srr were incubated in one of the above-mentioned buffers at 95°C for 15 min or 10 min, respectively, for the assay of racemase activity or dehydratase activity. (B) Temperature dependency of P. islandicum Srr activity. For the assay of racemase activity ({circ}), aliquots of 0.6 µg of Srr were incubated at various temperatures for 15 min in the presence of 400 mM L-serine and 10 µM PLP in 100 mM Tris-HCl buffer (pH 8.2). For the assay of dehydratase activity (•), aliquots of 0.6 µg of Srr were incubated at various temperatures for 10 min in the presence of 10 mM L-serine and 10 µM PLP in 100 mM Tris-HCl buffer (pH 7.8). Each plot represents the mean value of four independent experiments. The error bars show the standard deviations.

 
Basic enzymatic characteristics of Srr. The substrate specificity of the enzyme is shown in Table 3. The enzyme exhibited the highest racemase activity toward L-serine, followed by L-threonine, D-serine, and D-threonine. The dehydratase activity was also highest toward L-serine, followed by L-threonine. Both enantiomers of alanine, proline, aspartate, and glutamate were inert substrates of racemase, and the D-enantiomers of serine and threonine were also inert substrates of dehydratase. The Srr showed a Michaelis-Menten kinetic and produced straight lines in Lineweaver-Burk plots, giving Km and Vmax values for its racemase and dehydratase functions. A Hill plot analysis indicated that the dehydration reaction was not allosteric (data not shown). The Km, Vmax, and kcat values of the racemase and dehydratase activities are summarized in Table 4. The catalytic efficiency of racemase, kcat/Km, was 1.5-fold higher in the L- to D-serine conversion than in the reverse conversion. The kcat/Km value of dehydratase activity was 330 times as high as that of racemase activity when L-serine was used as the substrate.


View this table:
[in this window]
[in a new window]

  		TABLE 3. Substrate specificity of P. islandicum Srr

 

View this table:
[in this window]
[in a new window]

  		TABLE 4. Catalytic parameters of P. islandicum Srr as racemase and dehydratase

 
Effects of metal ions and L-isoleucine, L-valine, and adenyl nucleotides on the enzymatic activities of Srr. Metal ions (2 mM chloride salts), with the exception of Fe2+ and Cu2+, showed no significant effect on serine racemase or dehydratase activity (data not shown). Both enzymatic activities were decreased by 20 to 30% (P < 0.0001) in the presence of Fe2+ and increased by 30 to 40% (P < 0.0001) in the presence of Cu2+. Neither L-isoleucine nor L-valine showed any effects at 1 to 10 mM on these enzymatic activities (data not shown). Racemase activity was decreased modestly but significantly (P < 0.0003) by 10 to 20% by ATP at 0.1 to 1.0 mM, but not by ADP or AMP, when L-serine was used as the substrate in the presence of 2 mM MgCl2 (Fig. 6) or 2 mM CaCl2 (data not shown). The above analyses of variance were carried out using a Microsoft Excel 1-way analysis of variance test. The presence of 1 mM ATP did not affect the Lineweaver-Burk plots of the racemase reaction (data not shown). On the other hand, L-serine dehydratase activity was not affected by either of AMP, ADP, or ATP at 0.1 to 1.0 mM.


Figure 6
View larger version (28K):
[in this window]
[in a new window]

  		FIG. 6. Effects of ATP, ADP, or AMP on serine racemase (white bars) and serine dehydratase (shaded bars) activities of Srr in the presence of MgCl2. The values are expressed as percentages of the control without any nucleotides. The reaction mixture was composed of 0.6 µg of P. islandicum Srr, 100 mM Tris-HCl buffer (pH 8.2 for racemase assays and pH 7.8 for dehydratase assays), 10 µM PLP, 2.0 mM MgCl2, 400 mM (for racemase) or 10 mM (for dehydratase) L-serine, and either 0.1 mM or 1.0 mM of the indicated adenosine derivatives. The mean values of five independent experiments with standard deviations are shown.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Possible physiological function of Srr. Since Srr is a bifunctional enzyme, we examined the extent to which both enzymatic functions appear at the same time in the reaction vessel. In the present racemase activity asssay, the concentration of the substrate L-serine was 400 mM, more than twofold higher than the Km (185 mM). We calculated that after a 30-min reaction with 0.6 µg of Srr in a 200-µl reaction solution (pH 8.2), 0.36 µmol of D-serine and 1.15 µmol of pyruvate were produced, still leaving as much as 78.5 µmol (393 mM) of L-serine. In the dehydratase assay, 10 mM L-serine was incubated with the enzyme fraction for only 10 min, resulting in the production of 410 nmol of pyruvate. In the reaction (pH 7.8), the amount of D-serine produced was calculated to be only 4.7 nmol. Therefore, each enzyme was analyzed without being interfered with by the other enzyme's activity. Even under the optimum conditions for the racemization of serine, simultaneous production of three times more pyruvate than D-serine would be detected in the P. islandicum cells. Pyruvate may be used to drive the citric acid cycle (10) in cells grown heterotrophically in the direction that produces NADPH plus H+, NADH plus H+, FADH2, and ATP.

The dehydratase activity of Srr in P. islandicum cells also appears to be important because excess serine (of both enantiomers) is known to inhibit bacterial growth (5, 29), although the presence of this inhibition is unknown in archaea. The bacteriostatic effect of serine was suggested to be associated with inhibition of the biosyntheses of isoleucine and pantothenate (5, 29).

E. coli cells possess two well-studied allosteric threonine dehydratases. The biosynthetic enzyme, encoded by the ilvA gene, catalyzes the synthesis of L-valine from L-threonine, and the reaction is inhibited by L-isoleucine (28). The other, catabolic enzyme is encoded by the tdcB gene, and ATP synthesis by this enzyme is activated and inhibited by AMP and ATP, respectively (30). The fact that the enzymatic activities of Srr were not affected by these amino acids and nucleotides may indicate that Srr is not involved in the synthesis of either isoleucine or ATP, although ATP's weak inhibition of racemase activity may imply a more indirect relationship between racemase activity and ATP.

P. islandicum Srr and other, related enzymes. Studies of serine racemase are scarce among all organisms, while alanine racemase has been investigated in detail. The present study is one of only a few comprehensive works to cover the purification, gene cloning, and enzymatic characterization of a serine racemase. As far as we know, this is the first report on an archaeal serine racemase. The primary structure of Srr coincides completely with that of the putative threonine dehydratase of P. islandicum (CP000504; NC_008701), reported after our submission to the database. Srr exhibits 92.8% sequence similarity with the putative threonine dehydratases of P. aerophilum and Pyrobaculum calidifontis and around 66% similarity with those of several hyperthermophilic archaea (data not shown). Srr exhibits low similarity, less than 32%, with other serine racemases or serine/threonine dehydratases (Table 1). However, Srr possesses the amino acid sequence S-F-K-I-R-G, which is a highly conserved motif (6) among serine/threonine dehydratases from humans, rats, yeast, E. coli, etc., including the PLP-binding site of Srr, 53Lys (Fig. 3). Serine/threonine dehydratases are PLP dependent and widely distributed in nature. The glycine-rich region, 172Val to 185Val, is also highly conserved (6). Therefore, it has been revealed that important regions of the primary structure are conserved to reserve the enzyme functions of serine racemases and serine/threonine dehydratases of eukaryotes, bacteria, and archaea, although these enzymes possess a great variety of both primary and quaternary structures, yielding only moderate sequence homology with one another. These data may suggest that this family of enzymes arose from a common ancestor, which implies their necessity in all of these organisms. Consistent with this conjecture, the bacterial serine dehydratase is grouped in the class of "persistent" genes (7), which are dispensable for growth in a laboratory but are essential for survival under transition from one environmental condition to another and for population maintenance.

Srr resembles eukaryotic serine racemases (8, 19) rather than bacterial serine racemase (2), bacterial serine/threonine dehydratases, or homodimeric L-serine dehydratases from rat (23) and human (26) livers in that it is bifunctional. However, Srr is clearly different from both the eukaryotic enzymes (1, 22) and E. coli threonine dehydratases (28, 30) in that it is not an allosteric enzyme, and its enzymatic activity is not affected by Mg2+, Ca2+, or Mn2+ or, apparently, by nucleotides. Unlike the above-mentioned allosteric enzymes and the Arabidopsis serine racemase (8), Srr appears to be a homotrimer, based on the gel filtration (MW = 137K with the Shodex column; MW = 143K using light-scattering-refraction detection) and SDS-PAGE (MW = 43 to 44K) analyses. Of the many trimeric enzymes, only a branched-chain amino acid aminotransferase from E. coli is a PLP enzyme that forms a trimer of homodimers, as shown by a crystallographic study (24). In the enzyme, each monomer contains a PLP molecule.

Possible bifunctional mechanism of Srr. PLP per se, in the absence of enzyme, possesses weak serine racemization and dehydration activities by forming a Schiff base out of the amino group of serine and the formyl group on the pyridine nucleus of PLP (18, 27). In Srr, L-serine and the coenzyme PLP form a Schiff base that can alternatively react to yield either D-serine or pyruvate after the removal of H+ from the {alpha}-carbon of the L-serine; racemization takes place when H+ is added back to the {alpha}-carbon nonstereospecifically, while pyruvate is formed by dehydration when the hydroxyl group is removed from the L-serine (β-elimination) as H2O-yielding dehydroalanine that undergoes subsequent hydrolysis (4). The probability of the occurrence of racemization and dehydration may depend on the enzyme protein structure and the amino acid residues around the substrate and the Schiff base. Catalytic metals promote the formation of the Schiff base and stabilize it by chelation (18). Fe2+ at 1 mM enhanced the PLP-dependent nonenzymatic racemization two- to threefold (25) and increased the activity of Arabidopsis serine racemase by 40% (8), while in contrast, the ion inhibited Srr serine racemase activity by about 20%. This difference in the effect of Fe2+ on serine racemization may be ascribable to the structure of the protein surrounding the Schiff base in each enzyme.


   ACKNOWLEDGMENTS
 
We are grateful to Kihachiro Horiike and Tetsuo Ishida (Department of Biochemistry and Molecular Biology, Shiga University of Medical Science) for the analysis of Srr molecular weight by using the light-scattering photometer and refractometer.


   FOOTNOTES
 
* Corresponding author. Mailing address for Y. Nagata: Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-14 Kanda-Surugadai, Chiyoda Ward, Tokyo 101-8308, Japan. Phone: 81-3-3259-0861. Fax: 81-3-3293-7572. E-mail: nagata{at}chem.cst.nihon-u.ac.jp. Mailing address for S. Kasai (DNA cloning and sequencing questions): Department of Applied Chemistry and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, Sugimoto, Sumiyoshi, Osaka 558-8585 Japan. Phone and Fax: 81-6-6605-2783. E-mail: kasai{at}bioa.eng.osaka-cu.ac.jp Back

{triangledown} Published ahead of print on 26 October 2007. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Abe, K., S. Takahashi, Y. Muroki, Y. Kera, and R.-H. Yamada. 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.[Abstract/Free Full Text]
  2. Arias, C. A., M. Martin-Martinez, T. L. Blundell, M. Arthur, P. Courvalin, and P. E. Reynolds. 1999. Characterization and modeling of VanT: a novel, membrane-bound, serine racemase from vancomycin-resistant Enterococcus gallinarum BM4174. Mol. Microbiol. 21:1653-1664.
  3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef][Medline]
  4. Cordes, E. H., and W. P. Jencks. 1962. Semicarbazone formation from pyridoxal, pyridoxal phosphate, and their Schiff bases. Biochemistry 1:773-778.[CrossRef][Medline]
  5. Cosloy, S. D., and E. McFall. 1973. Metabolism of D-serine in Escherichia coli K-12: mechanism of growth inhibition. J. Bacteriol. 114:685-694.[Abstract/Free Full Text]
  6. Datta, P., T. J. Goss, J. R. Omnaas, and R. V. Patil. 1987. Covalent structure of biodegradative threonine dehydratase of Escherichia coli: homology with other dehydratases. Proc. Natl. Acad. Sci. USA 84:393-397.[Abstract/Free Full Text]
  7. Fang, G., R. Eduardo, and A. Danchin. 2005. How essential are nonessential genes? Mol. Biol. Evol. 22:2147-2156.[Abstract/Free Full Text]
  8. Fujitani, Y., N. Nakajima, K. Ishihara, T. Oikawa, K. Ito, and M. Sugimoto. 2006. Molecular and biochemical characterization of a serine racemase from Arabidopsis thaliana. Phytochemistry 67:668-674.[CrossRef][Medline]
  9. Hayashi, Y., H. Matsui, and T. Takagi. 1989. Membrane protein molecular weight determined by low-angle laser light-scattering photometry coupled with high-performance gel chromatography. Methods Enzymol. 172:514-528.[Medline]
  10. Hu, Y., and J. F. Holden. 2006. Citric acid cycle in hyperthermophilic archaeon Pyrobaculum islandicum grown autotrophically, heterotrophically, and mixotrophically with acetate. J. Bacteriol. 188:4350-4355.[Abstract/Free Full Text]
  11. Huber, R., J. K. Kristjansson, and K. O. Stetter. 1987. Pyrobaculum gen. nov., a new genus of neutrophilic, rod-shaped archaebacteria from continental solfataras growing optimally at 100°C. Arch. Microbiol. 149:95-101.[CrossRef]
  12. Kasai, S. 2006. Freshwater bioluminescence in Vibrio albensis (Vibrio cholerae biovar albensis) NCIMB 41 is caused by a two-nucleotide deletion in luxO. J. Biochem. 139:471-482.[Abstract/Free Full Text]
  13. Kasai, S., and T. Sumimoto. 2002. Stimulated biosynthesis of flavins in Photobacterium phosphoreum IFO 13896 and the presence of complete rib operons in two species of luminous bacteria. Eur. J. Biochem. 269:5851-5860.[Medline]
  14. Kasai, S., and T. Yamazaki. 2001. Identification of the cobalamin-dependent methionine synthase gene, metH, in Vibrio fischeri ATCC 7744 by sequencing using genomic DNA as a template. Gene 264:281-288.[CrossRef][Medline]
  15. König, H., and O. Kandler. 1979. The amino acid sequence of the peptide moiety of the pseudomurein from Methanobacterium thermoautotrophicum. Arch. Microbiol. 121:271-275.[CrossRef][Medline]
  16. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.[CrossRef][Medline]
  17. Marfey, P. 1984. Determination of D-amino acids. II. Use of a bi-functional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg Res. Commun. 49:591-596.
  18. Metzler, D. E., M. Ikawa, and E. E. Snell. 1954. A general mechanism for vitamin B6-catalyzed reaction. J. Am. Chem. Soc. 76:648-652.[CrossRef]
  19. Miranda, J. D., R. Panissutti, V. N. Foltyn, and H. Wolosker. 2002. Cofactors of serine racemase that physiologically stimulate the synthesis of the N-methyl-D-aspartate (NMDA) receptor coagonist D-serine. Proc. Natl. Acad. Sci. USA 99:14542-14547.[Abstract/Free Full Text]
  20. Nagata, Y., K. Tanaka, T. Iida, Y. Kera, R.-H. Yamada, Y. Nakajima, T. Fujiwara, Y. Fukumori, T. Yamanaka, Y. Koga, S. Tsuji, and K. Kawaguchi-Nagata. 1999. Occurrence of D-amino acids in a few archaea and dehydrogenase activities in hyperthermophile Pyrobaculum islandicum. Biochim. Biophys. Acta 1435:160-166.[CrossRef][Medline]
  21. Nagata, Y., K. Yamamoto, and T. Shimojo. 1992. Determination of D- and L-amino acids in mouse kidney by high-performance liquid chromatography. J. Chromatogr. 575:147-152.[Medline]
  22. Neidle, A., and D. S. Dunlop. 2002. Allosteric regulation of mouse brain serine racemase. Neurochem. Res. 27:1719-1725.[CrossRef][Medline]
  23. Ogawa, H., F. Takusagawa, K. Wakaki, H. Kishi, M. R. Eskandarian, M. Kobayashi, T. Date, N.-H. Huh, and H. C. Pitot. 1999. Rat liver serine dehydratase. J. Biol. Chem. 274:12855-12860.[Abstract/Free Full Text]
  24. Okada, H., K. Hirotsu, H. Hayashi, and H. Kagamiyama. 2001. Structure of Escherichia coli branched-chain amino acid aminotransferase and the complexes with 4-methylvalerate and 2-methylleucine: induced fit and substrate recognition of the enzyme. Biochemistry 40:7453-7463.[CrossRef][Medline]
  25. Olivard, J., D. E. Metzler, and E. E. Snell. 1952. Catalytic racemization of amino acids by pyridoxal and metal salts. J. Biol. Chem. 669:669-674.
  26. Sun, L., M. Bartlam, Y. Liu, H. Pang, and Z. Rao. 2005. Crystal structure of the pyridoxal-5'-phosphate-dependent serine dehydratase from human liver. Protein Sci. 14:791-798.[Abstract/Free Full Text]
  27. Tanigawa, M., N. Katagiri, H. Nakamura, and Y. Nagata. 2007. Nonenzymic racemization of amino acids catalyzed by pyridoxal 5'-phosphate at room temperature. Rep. Res. Inst. Sci. Technol. Nihon Univ. 49:1-9.
  28. Umbarger, H. E. 1973. Threonine deaminases. Adv. Enzymol Relat. Areas Mol. Biol. 37:349-395.[Medline]
  29. Uzan, M., and A. Danchin. 1978. Correlation between the serine sensitivity and the derepressibility of the ilv genes in Escherichia coli relA– mutants. Mol. Gen. Genet. 165:21-30.[CrossRef][Medline]
  30. Whanger, P. D., A. T. Phillips, K. W. Rabinowitz, J. R. Piperno, J. D. Shada, and W. A. Wood. 1968. The mechanism of action of 5'-adenylic acid-activated threonine dehydrase. J. Biol. Chem. 243:167-173.[Abstract/Free Full Text]
  31. Yamamoto, A., H. Tanaka, T. Ishida, and K. Horiike. 2007. Functional and structural characterization of D-aspartate oxidase from porcine kidney: non-Michaelis kinetics due to substrate activation. J. Biochem. 141:363-376.[Abstract/Free Full Text]

Journal of Bacteriology, February 2008, p. 1359-1365, Vol. 190, No. 4
0021-9193/08/$08.00+0     doi:10.1128/JB.01184-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Other Versions of this Article:
JB.01184-07v1
190/4/1359    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohnishi, M.
Right arrow Articles by Kasai, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohnishi, M.
Right arrow Articles by Kasai, S.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Appl. Environ. Microbiol. Infect. Immun. Eukaryot. Cell
Mol. Cell. Biol. J. Virol. Microbiol. Mol. Biol. Rev.
ALL ASM JOURNALS