An Aspartate Aminotransferase from an Extremely Thermophilic Bacterium, Thermus Thermophilus HB81

J. Biochem. 119, 135-144 (1996)

An Aspartate Aminotransferase from an Extremely Thermophilic Bacterium, Thermus thermophilus HB81

Akihiro Okamoto,*,2 Ryuichi Kato ,* Ryoji Masui,* Akihiko Yamagishi,t Tairo Oshima,t and Seiki Kuramitsu*,3 *Department of Biology , Faculty of Science, Osaka University, Toyonaka, Osaka 560; and tTokyo University of P harmacy and Life Science, Hachioji, Tokyo 192-03

Received for publication , August 30, 1995

The aspartate aminotransferase gene (AspAT , EC 2.6.1.1) of an extremely thermophilic b acterium, Thermus thermophilus HB8 , was cloned and sequenced, and its gene product was overproduced. The purified T. thermophilus AspAT was stable up to about 80•Ž at neutral pH. T. thermophilus AspAT was strictly specific for acidic amino acid substrates , such as aspartate, glutamate, and the respective keto acids . The gene coding for T. thermophilus AspAT showed that it comprised 1 ,155 by with a high G+C content (70 mol%), and encoded a 385-residue protein with a molecular weight of 42 ,050. The amino acid sequence of T. thermophilus AspAT deduced from its gene showed about 15 , 46, and 29%h omology with those from Escherichia coli, Bacillus sp. YM-2, and Sulfolobus solfataricus, respectively. When the amino acid sequence of T . thermophilus AspAT was compared with that of E. coli AspAT, the number of Cys was found to have decreased from 5 to 1, that of Asn from 23 to 9, that of Gln from 16 to 8, and that of Asp from 20 to 13 , all of which are known to be relatively labile at high temperatures. Conversely, the number of Pro was increased from 15 to 25, Arg from 22 to 32, and Glu 27 to 37. As shown by the E . coli AspAT structure, there was a marked tendency for the extra prolyl residues to be located around the surface of the molecule. This was quite different from that in the case of RecA

protein, which shows an increased number of prolyl residues in the interior of its molecule. Different strategies of different proteins as to prolyl contribution to thermostability have been suggested. Despite the high degree of conservation of active-site residues, Arg292 in E. coli AspAT, which interacts with the distal carboxylate of the substrate, was not found in T. thermophilus AspAT. Arg89 may complement the function of Arg292.

Key words: aspartate aminotransferase, gene cloning, prolyl residue, thermostability, Thermus thermophilus.

Pyridoxal 5'-phosphate (PLP) catalyzes a number of non directed mutagenesis (4): Lys258 is essential for donating enzymatic reactions, such as transamination, racemization, and abstracting a proton from the substrate (5), Tyr70 is decarboxylation, and ƒ¿,ƒÀ-elimination (1). The peptide necessary for recognizing C5 substrates, such as glutamate

portions of PLP-dependent enzymes select and promote a and 2-oxoglutarate (6), Tyr225 is necessary to preserve the specific non-enzymatic reaction, so high specific reactivity oxyanion form of PLP 0(3') (7, 8), Asp222 is necessary due and substrate specificity can be achieved. Aspartate amino to its negative charge but can be replaced by Glu (9), and transferase (AspAT) is the most intensively studied among His143 is not necessary, and a charge relay system either the PLP-dependent enzymes (2). The three-dimensional does not exist, or, if present, is not important for catalysis structures of the AspATs from various sources have been (10). The high specificity of AspAT for acidic amino acids is determined (3, the references therein). The roles of the attributed to the electrostatic interaction of Arg292 with active-site residues around PLP have been studied by site the distal carboxylate of the bound substrate. Although the aspartate and aromatic amino acid aminotransferases from 'This work was supported , in part, by Grants-in-Aid for Scientific Escherichia coli exhibit 44% amino acid sequence homology Research (Nos. 60153368, 06780502, and 06454654) from the and high activities toward acidic amino acid substrates, the Ministry of Education, Science and Culture of Japan, and by a grant from the Multidiscinlinarv Science Foundation. latter enzyme also exhibits high activity toward aromatic 2 Present address: Department of Medical Chemistry , Osaka Medical amino acid substrates (11). The substrate specificity of College, Takatsuki, Osaka 569. AspAT has also been studied by chimera analysis (12, 13). 3 To whom correspondence should be addressed. Tel: +81-6-850 In order to study the catalytic mechanism and substrate 5433, Fax: +81-6-850-5442, e-mail: [email protected], specificity of this enzyme, we prepared about twenty jp chimeras, some of which, however, were not stable under Abbreviations: AspAT, aspartate aminotransferase; bp, base pair; kb, kilobase pair; CD, circular dichroism; PCR, polymerase chain physiological conditions. Stable enzymes are necessary for reaction; PLP, pyridoxal 5'-phosphate; PMP, pyridoxamine 5'-phos further analysis of the catalytic mechanism of AspAT. phate. Chimera enzymes between thermophilic and mesophilic

Vol. 119, No. 1, 1996 135 136 A. Okamoto et al. ones may also provide us with information on the thermo (14), mixed oligonucleotide primers, 5'-GC(C/G)AC(C/G) stability of AspAT. Therefore, we cloned the AspAT gene GT(C/G)GC(C/G)GT(C/G)AA(C/G)GC-3' and 5'-AT(C/ from an extremely thermophilic bacterium, Thermus G)ACCTCGTC(C/G)CC(C/G/T)GG(G/A)TC-3', for the thermophilus HB8, which is a Gram-negative, aerobic, PCR (arrows in Fig. 1B) were synthesized using a MilliGen/ rod-shaped microorganism that can grow at temperatures Biosearch, Cyclone Plus DNA synthesizer. A DNA frag between 50 and 82•Ž, its optimum temperature being 75•Ž ment of about 300 by (the DNA fragment between the two (14). In this paper, we describe the gene cloning, sequenc arrows in Fig. 1B) was amplified by PCR, as described ing, expression in E. coli, and preliminary characterization previously (15), of AspAT from T. thermophilus HB8. Cloning and DNA Sequencing of the T. thermophilus AspAT Gene-In order to clone the entire T. thermophilus EXPERIMENTAL PROCEDURES AspAT gene, its genomic DNA was digested with various restriction endonucleases. Southern hybridization was Bacterial Strains and Materials-The DNA manipula carried out using the 32P-labeled PCR fragment and in the tion and bacterial cultivation procedures were similar to light of the results, we constructed mini DNA banks. One those described previously (15). E. coli strain AB1157 (16) comprised about 1.5kb XbaI-BglII fragments and the was used for overproduction of the T. thermophilus AspAT other of about 1.9kb BamHI-Kpnl fragments. These protein. The enzymes, reagents, and chromatographic fragments were ligated to the pUC119 vector through their materials used were purchased from commercial sources. respective restriction endonuclease sites. From these mini Amino Acid Sequence Determination The amino acid banks, two plasmids, pTG1 and pTG2 (Fig. 1A), were sequence was determined using an automated Edman obtained by colony hybridization. Since the lysates of the degradation protein sequencer (Applied Biosystems, model cells transformed with these plasmids showed no AspAT 473A). activity, it was suggested that these plasmids possessed Spectroscopy-The absorption spectra of the enzyme, at part of the T. thermophilus AspAT gene. Then, the pTG3 a protein concentration of about 15ƒÊM in a 1-cm cell, were plasmid, which possessed the entire gene region, was measured with a Hitachi spectrophotometer, model U constructed from pTG1 and pTG2 (Fig. IA). The nucleotide 3000. The buffer solution comprised 100mM KCl, 10ƒÊM sequence of the HindIII-Xhol region of pTG3 was deter EDTA, and a buffer component of 50mM acetate (pH 4.6 to mined on both strands. The DNA fragments were sub 5.0), 50mM phosphate (pH 5.5 to 7.4), or 50 mM HEPES cloned into the M13mp18 and M13mp19 phages, single (pH 8.0). stranded DNAs were prepared using E. coli AK101 and The circular dichroic (CD) spectra of AspAT, at a protein helper phage M13K07 (15, 20), and DNA sequence anal concentration of about 0.1mg/ml in a 1-mm cell, were yses were performed by the dideoxy termination method measured with a Jasco spectropolarimeter, model J-500A. (21) using the Taq cycle sequencing system and an Applied The buffer used comprised 100mM KC1, 10ƒÊM EDTA, Biosystems model 373A DNA sequencer. and a buffer component of 10mM borate and 15mM Purification of Recombinant T. thermophilus AspAT phosphate (pH 8.0). Thermal stability was measured at T. thermophilus AspAT was overproduced in E. coli 222nm with a heating rate of 1•Ž/min. AB1157 cells transformed with pTG3 in a medium com Catalytic Activity Assays-The overall transamination prising 0.5% yeast extract (Nacalai Tesque, Kyoto), 0.8% activity was measured spectrophotometrically at 340nm Polypepton (Nippon Shinyaku, Tokyo), and 0.25% NaCl by a coupled assay with malate dehydrogenase and NADH (pH 7.2-7.4). The direction of the T. thermophilus AspAT at pH 8.0 and 25•Ž (17), and the steady-state kinetic gene was the same as that of the lac promoter of pUC119. parameters, Km and lat, were determined (18, 19). The The transformed AB1157 was cultivated overnight, and the reaction mixtures comprised 50mM HEPES, 100mM cells were harvested and stored at -20•Ž until use. KCl, 2.4 units/ml malate dehydrogenase, 0.15mM NADH, All the following operations, except heat treatment, were 10ƒÊM PLP, about 15nM AspAT, and various concentra carried out at 4•Ž. Wet cells (48 g from 12 liters culture) tions of L-aspartate or 2-oxoglutarate. were suspended in 25mM Tris buffer containing 50mM The half-transamination reaction of the PLF form o1 glucose and 10mM EDTA (pH 8.0), and then disrupted by AspAT with L-phenylalanine or L-alanine was followed by sonication. The disrupted cells were diluted with 500ml of monitoring of the changes in the absorbance of the bound a solution (pH 8.0) comprising 10mM 2-oxoglutarate, 50ƒÊ PLP at 380nm, pH 8.0 and 25•Ž. The reaction solutions M PLP, 10mM 2-mercaptoethanol, and 1mM EDTA, and comprised 50mM HEPES, 100mM KCl, 20ƒÊM PLP, 15 then heat-treated by incubation at 70•Ž for 20 min. After ƒÊ M T. thermophilus AspAT, and various concentrations of centrifugation at 8,000rpm for 20 min, the supernatant L-phenylalanine or L-alanine. was adjusted to pH 8.5 by adding 1M potassium carbonate Design of Oligonucleotide Primers and Internal Gene buffer, and then loaded onto a SuperQ-Toyopearl 650M Fragment Amplification by the Polymerase Chain Reac column (bed volume, 250ml) equilibrated with 10mM tion-First, we purified T. thermophilus AspAT from T. potassium carbonate (pH 8.5) containing 5mM 2-mercap thermophilus HB8 using procedures similar to those used toethanol, 5mM succinate, and 1mM EDTA. The column for E. coli AspAT (19). The purified protein was digested was washed with 2 bed-volumes of the equilibration buffer with lysylendopeptidase, and the resulting peptide frag and then eluted with a linear gradient of 0-0.5M NaCl in ments were separated by high-pressure liquid chromatog the same buffer (total volume, 1 liter). The active fractions raphy. The amino acid sequences of the four separated were pooled, solid ammonium sulfate (final saturation, peptide fragments and the native whole protein were 75%) was added, and the resulting precipitate was collected determined. On the basis of the amino acid sequences and by centrifugation and dialyzed against dialysis buffer (pH the high G+C content of T. thermophilus genomic DNA 6.5) comprising 50mM potassium phosphate, 0.8M

J. Biochem. Aspartate Aminotransferase from Thermus thermophilus 137

Fig. 1. Cloned DNA fragments, and the nucleotide and amino the XhoI region of pTG3 is shown. The translation, using the single acid sequences of the T. thermophilus AspAT gene. (A) The letter code, is given below the nucleotide sequence. Amino acid cloned DNA fragments (pTG1 and pTG2) and constructed plasmid sequences confirmed by peptide sequencing are underlined. Dotted (pTG3) are shown by lines. The boxed arrow indicates the T. thermo underlines mean ambiguous identification. The two regions used as PCR primers are indicated by arrows. The nucleotide sequence data philus AspAT gene. The restriction enzyme sites are as follows: B, BamHI; Bg, BglII; H, HindIII; K, KpnI; N, Ncol; Xb, XbaI; X, Xhol. reported in this paper appear in the GSDB, DDBJ, EMBL, and NCBI (B; e nucleotide sequence between 27 by upstream of HindIIl and nucleotide sequence databases under the accession number , D38459.

9, No. 1, 1996 138 A . Okamoto et al.

ammonium sulfate, 5mM succinate , 5mM 2-mercaptoeth from 23 to 9, that of Gln from 16 to 8, and that of Asp from anol, and 1mM EDTA. The dialyzate was loaded onto a 20 to 13, all of which are known to be relatively labile at Phenyl-Toyopearl 650M column (bed volume, 100ml) high temperatures (23-27). Conversely, the number of Pro equilibrated with the dialysis buffer, washed with 2 bed residues increased from 15 to 25, that of Arg from 22 to 32, volumes of the same buffer, and then eluted with a linear and that of Glu from 27 to 37. The two prolyl residues in the gradient generated from 500ml of dialysis buffer alone to cis conformation (3) were conserved. The amino acid 500ml of a buffer (pH 8.0) comprising 25% (v/v) ethylene composition of T. thermophilus AspAT suggests that its glycol, 7.5mM HEPES buffer, 5mM succinate, 5mM isoelectric pH value is about 0.5-pH units higher than that 2-mercaptoethanol, and 1mM EDTA (total volume, 1 of E. coli. The more basic nature of T. thermophilus AspAT, liter). Solid ammonium sulfate (final saturation, 75%) was as compared with E. coli AspAT, was confirmed by their added to the pooled active fractions, and the resulting behaviors on anion exchange column chromatography (data precipitate was collected by centrifugation and dialyzed not shown). against 10mM HEPES buffer (pH 8.0) containing 100mM The amino acid composition of T. thermophilus AspAT KC1, 5mM succinate, 5mM 2-mercaptoethanol, and 1mM differed slightly from that of T. aquaticus AspAT deter EDTA. The dialyzate was loaded onto a Sephacryl S-200 mined by amino acid analysis (22). For example, T. column (bed volume, 400ml) equilibrated with the dialysis thermophilus AspAT has only one cysteinyl residue at buffer. The pooled active fractions were mixed with twice position 280,4 whereas T. aquaticus AspAT has no cysteinyl the volume of glycerol and then stored at -20•Ž. residue. Such a difference in protein sequences derived Determination of the Protein Concentration-The con from these two thermophiles has been suggested by analy centration of the PLP form of T. thermophilus AspAT was sis of restriction sites in their genomic DNAs (28). This is determined spectrophotometrically using a molar extinc also the case for 3-isopropylmalate dehydrogenase (29), tion coefficient, EM, of 3.55 x 104cm-1 • M-1 at 280nm and elongation factor Tu (30, 31), and RecA protein (15, 32, pH 8.0 (19). 33). Expression of the T. thermophilus AspAT Gene in E. RESULTS coli-To prepare a large amount of T. thermophilus AspAT, E. coli AB1157 was transformed with plasmid pTG3 (Fig. Cloning and DNA Sequencing of the T. thermophilus 1A), which possessed the entire T. thermophilus AspAT AspAT Gene-As described under "EXPERIMENTALPRO gene. Although this E. coli strain possesses its own AspAT, CEDURES", we first purified the AspAT enzyme from T. its activity was abolished by heat treatment during puri thermophilus HB8, digested the purified protein with fication. The purification results are summarized in Table I. lysylendopeptidase, and then determined the amino acid The purified recombinant enzyme gave a single band on a sequences of the four separated peptide fragments as well polyacrylamide gel electrophoresis in the presence of as the native whole protein (underlined in Fig. 1B). On the sodium dodecyl sulfate and 2-mercaptoethanol, and its basis of the amino acid sequences, the genomic AspAT gene molecular weight was estimated to be about 42,000, which was cloned from T. thermophilus HB8 (pTG3 in Fig. 1A). is consistent with that predicted from the DNA sequence. The nucleotide sequence of pTG3 was determined, as The N-terminal amino acid sequence of this recombinant shown in Fig. 1B. The T. thermophilus AspAT gene com enzyme was identical to that of the native enzyme (data not prised 1,155 by with an ATG initiation codon and a TAG shown). These results confirmed that the recombinant termination codon. The G+C content of the open reading protein expressed in E. coli was T. thermophilus AspAT. frame was 70 mol%, which is similar to the genomic G+C Spectrophotometric Properties of the Enzyme-Bound content (69 mol%) of T. thermophilus HB8 (14). This open PLP-The absorption maximum of the recombinant pro reading frame encodes a 385-residue protein with a tein was 430nm at acidic pH and shifted to 380nm at predicted molecular weight of 42,050. The amino acid alkaline pH (Fig. 2). The EM value at acidic pH was 7,100 sequences of the peptide fragments obtained on lysylen cm-1•œM-1 at 430nm, and that at alkaline pH was 5,400 dopeptidase digestion (underlined amino acid residues in cm1-M-1 at 380nm. The deprotonated format alkaline pH Fig. 1B) were identical to those predicted from the DNA showed a slightly different absorption spectrum from sequence. The N-terminus of T. thermophilus AspAT was vertebrate and E. coli AspATs, which exhibit absorption not blocked, unlike in the case of T. aquaticus AspAT, maxima at 360nm (34). The pH-dependent EM values at which has a blocked N-terminus (22). 380 and 430nm indicated identical pKa values of 6.1 (inset When the amino acid sequence of T. thermophilus AspAT in Fig. 2). This pKa value was slightly lower than the values was compared with that of E. coli AspAT, the number of for vertebrate and E. coli AspATs (34). Conversion of the Cys was found to have decreased from 5 to 1, that of Asn PLP form of the enzyme into the pyridoxamine 5'-phos

TABLE I. Purification of recombinant T. thermophilus AspAT from E. coli.

4The amino acid residues are numbered according to the sequence of porcine cytosolic aspartate aminotransferase (51).

J. Biochem. Aspartate Aminotransferase from Thermus thermophilus 139

Fig. 2. Effect of pH on absorption spectra of the PLP-form enzyme and absorption spectrum of the PMP-form enzyme. Absorption spectra of the PLP-form enzyme at the several pHs indicated in the figure are shown by solid lines and that of the PMP-form at pH 8.0 by a broken line. Changes of the apparent molar absorp tion coefficients for the PLP-form enzyme against pH at 380nm (•›) and 430nm (•¢) are plotted in the inset. The solid lines in the inset are the theoretical curves with a pKa value of 6.1.

dent spectral change in the visible absorption region, and its spectra and Schiff base pKa value are known to reflect the microenvironment around the PLP (34). Therefore, these results suggest that the microenvironment around the PLP in T. thermophilus AspAT is slightly different from that in the E. coli enzyme. The gross conformation and thermostability of T. ther mophilus AspAT were examined by CD spectroscopy. The CD spectrum of T. thermophilus AspAT in the region between 200 and 250nm (Fig. 3A) exhibited negative double minima at 208 and 222nm, characteristic of a-helical structures. The thermostability of T. thermo philus AspAT was estimated by measuring the change in the ellipticity at 222nm (Fig. 3B). No thermal denatura tion of T. thermophilus AspAT was observed below 75•Ž, whereas at 49•Ž, E. coli AspAT was denatured. Thermal denaturation of T. thermophilus AspAT was irreversible, as is the case for E. coli AspAT. Catalytic Properties-The overall transamination reac tion of AspAT proceeds via the ping-pong bi-bi mechanism. The steady-state kinetic parameters of the overall reac tions with L-aspartate and 2-oxoglutarate were determined at 25, 35, and 45•Ž. The respective values were: kmax, 110, 210, and 270 s-1, and Km, 1.8, 1.5, and 1.1mM for L aspartate, and Km, 2.3, 1.7, and 1.0mM for 2-oxoglutarate. In comparison with its high activity toward L-aspartate, Fig. 3. Far-UV CD spectrum of T. thermophilus AspAT. (A) CD the PLP form of T. thermophilus AspAT showed no activity spectrum of the PLP-form enzyme. The buffer solution comprised 100 toward hydrophobic amino acids (data not shown). mM KCI, 10ƒÊM EDTA, and a buffer component of 10mM borate and 15mM phosphate (pH 8.0). The measurement was performed at 25•Ž. (B) Thermal -unfolding curves for AspAT isolated from T. DISCUSSION thermophilus (continuous line) and E. coli (broken line). Mean residue ellipticities at 222 nm are plotted against temperature. The buffer Sequence Alignment of AspATs-AspATs were classifi solution was the same as in the legend to A. ed into aminotransferase subgroup I, according to Mehta et al. (35), except AspAT from the archaebacterium, Meth anobacterium thermoformicicum strain SF-4, which was phate (PMP) form with cysteine sulfinate shifted the classified into subgroup IV (36). The AspATs in subgroup I absorption maximum to 330nm (broken line in Fig. 2), are further subdivided, on the basis of their amino acid with an em value of 6,800 cm-1 -M-1. In the case of E. coli sequences, into two subgroups: one subgroup comprises AspAT, the coenzyme PLP forms an internal Schiff base AspATs isolated from E. coli, yeast, chicken , pig and so on, with the catalytic Lys258 residue in the absence of sub and the other those isolated from the thermophilic bacter strate. This coenzyme induces a characteristic pH-depen ium, Bacillus sp. YM-2 (37), and the thermoacidophilic

Vol. 119, No. 1, 1996 140 A. Okamoto et al. archaebacterium, Sulfolobus solfataricus (38). We call subgroup Ib AspATs. Figure 5 shows their amino acid these subgroups subgroups Ia and lb. The amino acid compositions, which differ markedly. There were fewer sequence homologies between subgroups la and lb are very Asn in T. thermophilus than Bacillus sp. YM-2 and S. low (about 15%), but the active-site residues are well solfataricus AspATs, whereas the number of Pro showed conserved. the opposite tendency (Fig. 5A). As judged on the alignment Figure 4 compares the amino acid sequence of T. thermo of the sequences, the Ile residues in Bacillus sp. YM-2 and philus HB8 AspAT with those of Bacillus sp. YM-2, S. S. solfataricus AspATs are replaced by Val and Leu in T. solfataricus, E. coli, and pig cytosolic AspATs. T. thermo thermophilus AspAT, respectively, and there are less Ile philus AspAT showed 46% homology with Bacillus sp. than Val or Leu in T. thermophilus AspAT (Fig. 5B). Lys in YM-2 AspAT and 29% with S. solfataricus AspAT, but 16 Bacillus sp. YM-2 and S. solfataricus AspATs was replaced and 10% with E. coli and pig cytosolic AspATs, respective by Arg in T. thermophilus AspAT (Fig. 5C). There was no ly. These results indicate that T. thermophilus AspAT Cys in Bacillus sp. YM-2 and S. solfataricus AspATs, and belongs to subgroup Ib. We also compared the amino acid only one in T. thermophilus AspAT. The implication of composition of T. thermophilus AspAT with those of other these results will be discussed later.

Fig. 4. Aligned amino acid sequences of AspATs. Gaps (-) have been inserted to achieve maximum homology. White letters on black indicate the residues conserved in T. thermophilus AspAT and other aligned AspATs. Black letters on gray indicate similar residues in T. thermophilus AspAT and other aligned AspATs. Similar residues are grouped as follows: D, E; S, T; I, L, V, M; F, W, Y; N, Q; H, R, K; A; G; C; P. Asterisks (*) indicate the active site residues in E. coli AspAT. The numerical sign (#) indicates Arg292. The abbreviations of the sources of the enzymes are: T.th., Thermus thermo philus HB8; B.YM-2, Bacillus sp. YM-2; S.sol., Sulfolobus solfataricus; E.coli, Escherichia coli; cPig, pig cytosol.

J. Biochem. Aspartate Aminotransferase from Thermus thermophilus 141

Active Site of T. thermophilus AspAT-Although T. the case of E. coli. Bacillus sp. YM-2 AspAT, which thermophilus AspAT exhibited very low homology (16%) exhibited high sequence homology with T. thermophilus with E. coli AspAT, the active site residues in E. coli AspAT at the active site, showed absorption peaks at 360 AspAT (i.e. Tyr70, G1y107, Trp140, Asn194, Asp222, and 415nm, and the pKa value of its internal aldimine Tyr225, Lys258, Arg266, and Arg386) are well conserved nitrogen was 5.7 (39). These spectroscopic differences in T. thermophilus AspAT, as they are in Bacillus sp. YM-2 suggest that AspATs from different sources differ slightly and S. solfataricus AspATs. Despite the high degree of from each other in the microenvironment around the PLP, conservation of these active-site residues, Arg292 in E. coli which may lead to the varying substrate specificities of AspAT, which interacts with the distal carboxylate of the these enzymes. bound substrate (3), was not found in T. thermophilus Thermostability-Thermal unfolding experiments in AspAT, as is the case for AspATs from Bacillus sp. YM-2 dicated that T. thermophilus AspAT is more thermostable (37) and S. solfataricus (38). The high specificity for than the E. coli enzyme. This thermostability should be dicarboxylate substrates of T. thermophilus AspAT in related to its amino acid composition. Menendez-Arias and comparison with E. coli AspAT may be attributed to the Argos (40) compared the amino acid sequences of six residue responsible for recognition of the distal carbox different protein families, each of which consists of both ylate. thermophilic and mesophilic enzymes, and includes at least Which residue in these AspATs contributes to the one enzyme of which the tertiary structure has been recognition of the substrate, carboxylate? The following determined. They reported that alterations in protein points should be considered: (i) The residue will be Arg, flexibility and hydrophobicity provided the main driving Lys, or His, because it interacts with the negatively force for achieving thermal stability. Some of the amino charged carboxylate of the substrate. (ii) The residue is acid substitutions observed in T. thermophilus AspAT, expected to be conserved in T. thermophilus, Bacillus sp. described under "RESULTS," can be explained in this way. YM-2, and S. solfataricus AspATs, as the corresponding Furthermore, thermostable enzymes may be constructed residues, except Arg292, in the active sites are well to prevent the inactivation that occurs with aging, which conserved. Arg89 (Arg90 in S. solfataricus AspAT), was found to be caused by post-translocational modification Lys109, Lys184, and Arg329 satisfy both conditions (i) and (deamidation, isomerization, racemization, carbamoyla (ii). Since Arg292 of E. coli AspAT is situated in the tion, oxidation, and so on) (41). Deamidation of Asn is one conserved region in the C-terminal domain, the residues in of the most prevalent modifications. Carbamoylation, the the vicinity of the alternative residue are also expected to reaction of isocyanate with the E -amino group of Lys, and be conserved, especially among the AspATs from thermo isomerization of the ƒ¿-Asp-Gly sequence to ƒÀ-Asp-Gly philes. Therefore, Arg89 and Arg329 are the prime candi have also been observed (26). Thus, reductions in the dates. According to the X-ray crystallographic results for numbers of Asn and Lys residues may protect an enzyme AspATs in subgroup I, Arg329 on the same subunit is against irreversible inactivation. located in the middle of the longest a -helix, which bridges A reduced Cys level was also observed in T. thermophilus the large and small domains, and is situated a little apart AspAT, as well as other AspATs from thermophilic bacte from the active site. Arg89 is located closer to the active ria. Covalent crosslinking by cysteine residues can be site than Arg329 on the other subunit. On the basis of the utilized to stabilize T4 lysozyme (42). However, the location of Arg292, it is possible to suppose that the residue proteins in cells cannot use the disulfide bond strategy, responsible for recognition of the substrate, carboxylate, is because the cellular interior is a reductive environment. supplied by the other subunit of the dimer. Therefore, it The reduction of the Cys level may contribute to enzyme seems more likely that in T. thermophilus AspAT, Arg89 thermostability by protection of the enzyme against ir complements the function of Arg292. reversible inactivation, since the sulfhydryl group is liable The absorption spectra and Schiff base pKa value of T to be modified (23). thermophilus AspAT were slightly different from those in Another characteristic of T. thermophilus AspAT is the

Fig. 5. Comparison of the amino acid compositions of AspATs belonging to subgroup lb. The compositions of (A) Asn and Pro, (B) Ile, Val, and Len, and (C) Arg and Lys are indicated. Ordinates show the number of residues in a subunit.

Vol. 119, No. 1, 1996 142 A. Okamoto et al.

Fig. 6. Distribution of the resi dues substituted by proline in T. thermophilus AspAT (A) and RecA protein (B). The models for the struc tures of these proteins were derived from the crystal structures of the E. coli counterparts. Images were pro duced from 1ARS (3) and 2REB (52) of the Brookhaven Protein Data Bank (53) using the program, RasMol2. The residues substituted by proline in the T. thermophilus proteins are repre sented as red spheres. The a-helices and 8-sheets are violet and green, respectively. Other regions are gray. In (A) one subunit of the dimer is colored.

unusually high Pro content (25 residues) compared with that of E. coli AspAT. This can be rationalized by the mesophilic AspATs, such as E. coli AspAT (15 residues). existence of many proteins which show significant similar Compared with its mesophilic counterparts, high Pro ities in their backbone conformations, despite not showing contents have been found in many proteins from T. thermo high sequence similarities (49). philus, such as 3-isopropylmalate dehydrogenase (29), Surprisingly, most of the residues substituted by proline seryl-tRNA synthetase (43), aspartyl-tRNA synthetase in T. thermophilus AspAT were distributed predominantly (44), manganese superoxide dismutase (45), and RecA on the surface regions of the molecule, rather than in its protein (15). The importance of Pro residues for thermo hydrophobic core (Fig. 6A). These proline residues are not stability has been demonstrated by site-directed muta clustered in a specific region but are widely distributed over genesis (46-48). It is thought that, theoretically, such a the surface of the large domain of the molecule. The substitution reduces conformational entropy in the unfold preferred distribution of proline residues on the molecular ed state, which can contribute to protein stabilization. surface was also observed for 3-isopropylmalate dehy Detailed studies have shown that a Pro residue contributes drogenase (29), aspartyl-tRNA synthetase (44), and to protein stabilization to varying extents, depending on the manganese superoxide dismutase (45). Contrary to these features of the local structure where the residue is located proteins, the residues substituted by proline in T. thermo in the protein (47, 48). Therefore, it is necessary to philus RecA protein (15) were located in a relatively compare the tertiary structures of the proteins from narrow region (Fig. 6B). These replacement biases might mesophiles and thermophiles to reveal the importance of be related to the dynamic motion of the protein molecules. the high Pro contents of thermostable proteins. One explanation already discussed is that the substitu The tertiary structure of E. cola AspAT has been deter tion of Pro for other amino acids reduces the confor mined, but that of T. thermophilus AspAT is unknown. mational entropy in the unfolded state and increases the Although the primary structures of T. thermophilus and E. free energy of the denatured state. This increases the coli AspATs showed relatively low homology, their N-ter relative free energy difference between the native and minal regions containing approximately 250 residues denatured states, which contributes to the thermal stabili showed a relatively higher degree of similarity than their zation of the protein. In particular, the introduction of Pro other regions. In particular, their active-site residues were residues into a bending region, the conformation of which is well conserved, as discussed above. Therefore, it is conceiv inclined to be random, could restrict fluctuations in such a able that the backbone conformation of T. thermophilus region. Another possibility is that substitution by proline AspAT is, at least in the N-terminal region, equivalent to increases the conformational enthalpy in the native state,

J. Biochem. Aspartate Aminotransferase from Thermus thermophilus 143

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