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J Biol Chem, Vol. 273, Issue 45, 29554-29564, November 6, 1998

The Novel Substrate Recognition Mechanism Utilized by Aspartate Aminotransferase of the Extreme Thermophile Thermus thermophilus HB8^*

Yuko Nobe, Shin-ichi Kawaguchi, Hideaki Ura, Tadashi Nakai^§, Ken Hirotsu^§, Ryuichi Kato, and Seiki Kuramitsu^¶

From the  Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan and the ^§ Department of Chemistry, Graduate School of Science, Osaka City University, Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan

	ABSTRACT

Top Abstract Introduction Materials & Methods Results Discussion References

Aspartate aminotransferase (AspAT) is a unique enzyme that can react with two types of substrate with quite different properties, acidic substrates, such as aspartate and glutamate, and neutral substrates, although the catalytic group Lys-258 acts on both types of substrate. The dynamic properties of the substrate-binding site are indispensable to the interaction with hydrophobic substrates (Kawaguchi, S., Nobe, Y., Yasuoka, J., Wakamiya, T., Kusumoto, S., and Kuramitsu, S. (1997) J. Biochem. (Tokyo) 122, 55-63). AspATs from various organisms are classified into two subgroups, Ia and Ib. The former includes AspATs from Escherichia coli and higher eukaryotes, whereas the latter includes those from Thermus thermophilus and many prokaryotes. The AspATs belonging to subgroup Ia each have an Arg-292 residue, which interacts with the distal carboxyl groups of dicarboxylic (acidic) substrates, but the functionally similar residue of subgroup Ib AspATs has not been identified. In view of the x-ray crystallographic structure of T. thermophilus AspAT, we expected Lys-109 to be this residue in the subgroup Ib AspATs and constructed K109V and K109S mutants. Replacing Lys-109 with Val or Ser resulted in loss of activity toward acidic substrates but increased that toward the neutral substrate, alanine, considerably. These results indicate that Lys-109 is a major determinant of the acidic substrate specificity of subgroup Ib AspATs. Kinetic analysis of the interactions with neutral substrates indicated that T. thermophilus AspAT is subject to less steric hindrance and its substrate-binding pocket has a more flexible conformation than E. coli AspAT. A flexible active site in the rigid T. thermophilus AspAT molecule may explain its high activity even at room temperature.

	INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Aminotransferases are well known pyridoxal 5'-phosphate (PLP¹)-dependent enzymes that catalyze the reversible transfer of the amino group from an amino acid to a 2-keto acid (1-5),

(Eq. 1)

where E_L and E_M denote the PLP and pyridoxamine 5'-phosphate (PMP) forms of the enzyme, respectively. A number of aminotransferases with different substrate specificities have been identified, and their amino acid sequences have been reported. Aminotransferases are classified into four subgroups (I-IV), which, with exception of family III, are thought to have a common evolutionary origin (6). Subgroup I includes aminotransferases that use aspartate, alanine, histidine, or an aromatic amino acid as the amino group donor, and many aminotransferases of this subgroup utilize common dicarboxylic acids (such as 2-oxoglutarate and glutamate). Under physiological conditions, aspartate aminotransferase (AspAT) uses aspartate and glutamate as amino group donors and oxaloacetate and 2-oxoglutarate as amino group acceptors. The amino acid sequences of AspATs from eubacteria, archaebacteria, and eukaryotes have been reported, and AspATs are divided into subgroups Ia and Ib according to their mutual homologies (7-9). To date, subgroup Ia includes the AspATs from eubacteria and eukaryotes, and subgroup Ib includes those from eubacteria and archaebacteria. The amino acid sequence identities between members of subgroup Ia are higher than 40%; identities between subgroup Ib are 30-40%. However, the identities are only 15% when the subgroup Ia and Ib members are compared (8). Subgroup Ia AspATs have been more widely and extensively investigated (Ref. 10 and references therein) than subgroup Ib AspATs and other aminotransferases with different substrate specificities. However, subgroup Ib AspATs from Sulfolobus solfataricus (11), Bacillus sp. strain YM-2 (12), and Thermus thermophilus HB8 (8) have been characterized physicochemically in vitro.

T. thermophilus is an extremely thermophilic eubacterium that can grow at temperatures up to 85 °C (its optimum growth temperature is 75 °C) (13). The amino acid sequence of T. thermophilus AspAT deduced from its gene (8) revealed that T. thermophilus AspAT shows 89% sequence identity with Thermus aquaticus YT1 AspAT (14), and 30-45% identities with subgroup Ib AspATs from S. solfataricus (15), Bacillus sp. strain YM-2 (16), and Rhizobium meliloti (7, 17), but only 15% sequence identifies with the subgroup Ia AspATs. Therefore, T. thermophilus AspAT is a member of the subgroup Ib AspATs. In common with other enzymes from thermophilic organisms, T. thermophilus AspAT is stable at temperatures up to 80 °C, and its high thermostability is thought to be related to its amino acid composition (8). The high Pro content of T. thermophilus AspAT will make the enzyme rigid and thermostable, and its low content of heat-labile amino acid residues (Asn, Lys, and Cys) may prevent aging and irreversible inactivation. However, unlike T. thermophilus 3-isopropylmalate dehydrogenase (18), the reactivity of T. thermophilus AspAT is nearly equal to those of AspATs from mesophilic organisms at the same room temperature (25 °C) (8). Such enzymatic adaptation for a wide range of temperatures may generate interest for biotechnological applications.

Studies on the three-dimensional structures of subgroup Ia AspATs from Escherichia coli and vertebrates revealed that their spacial structures are essentially the same and demonstrated many functional residues (Tyr-70, Asn-194, Pro-195, Gly-197, Asp-222, Tyr-225, Lys-258, Arg-266, Arg-292, and Arg-386)² in their active sites (19-26). These functional residues are almost completely conserved, even in the AspATs of subgroup Ib. The residue that interacts directly with a distal carboxyl group of an acidic substrate, which is the Arg-292 residue in the subgroup Ia AspATs, has not been identified in the primary structure of subgroup Ib AspATs, which are, of course, highly active toward acidic substrates.

Very recently, we succeeded in crystallizing T. thermophilus AspAT and determining the three-dimensional structure of its substrate-free form (Fig. 1a, Protein Data Bank file 1BKG) (27). The structure of this enzyme led us to propose that Lys-109 is the residue that recognizes a distal carboxyl group of a dicarboxylic substrate because the distal amino group of the Lys-109 side chain is located near the position equivalent to that of Arg-292 in the structure of subgroup Ia AspATs complexed with an acidic substrate (Fig. 1c), and this Lys residue is well conserved among subgroup Ib members (Fig. 2), whereas subgroup Ia AspATs have a Thr residue at this position (previously, we suggested that Arg-89 might recognize the distal carboxyl group of the substrate (8), but Arg-89 is not conserved among subgroup Ib aminotransferases). Therefore, we constructed mutant enzymes of T. thermophilus AspAT by replacing Lys-109 with other residues and analyzed their substrate specificities to establish whether Lys-109 is a direct determinant of the acidic substrate specificity of subgroup Ib AspATs.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Structure of the active site of aspartate aminotransferase. a, T. thermophilus AspAT in the substrate-free form (27). b, E. coli AspAT in the substrate-free form (22). c, E. coli AspAT complexed with 2-methylaspartate, acidic substrate analog (22). Single letter codes are used for amino acid residues.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   The amino acid sequence alignment of subgroup Ib aspartate aminotransferase in the region from the 50th to 110th residue. The numbers shown to be left and right of the sequences are the amino acid numbers of the beginning and end of each sequence segment, respectively. Tyr-70 (Y70), which is conserved in subgroup I aminotransferases, is indicated by the arrow. Lys-109 (K109) is indicated by an arrow and a box. The amino acid residues of Tyr-70 and Lys-109 are numbered according to the sequence of pig cytosolic aspartate aminotransferase (62). * and : show residues that are conserved and homologous among all entries, respectively. Abbreviations: Tth, T. thermophilus HB8 AspAT (8); Taq, T. aquaticus YT1 AspAT (14); RmeA, Sinorhizobium meliloti AspAT-A (17); RmeB, Sinorhizobium meliloti AspAT-B (7); Svi, Streptomyces virginiae AspAT (63); Bst, Bacillus stearothermophilus AspAT (64); Bsu, B. subtilis AspAT (61); Bsp, Bacillus sp. strain YM-2 AspAT (16); Ssp, Synechocystis sp. strain PCC6803 putative AspAT; Hpy, Helicobacter pylori putative AspAT (65); Afu, Archaeoglobus fulgidus putative AspAT (66); Sso, S. solfataricus AspAT (15).

AspATs show catalytic activities toward both acidic and neutral substrates (5, 28-30). E. coli AspAT (subgroup Ia) has been found to show higher reactivities with neutral substrates than other AspATs (5) studied. Previous studies indicated that the subgroup Ib AspATs show high acidic substrate specificities and only react weakly with neutral substrates (8, 11, 12). However, replacement of Lys-109 of T. thermophilus AspAT with a neutral amino acid residue resulted in loss of activity toward acidic substrates but increased that toward hydrophobic substrates markedly.

	MATERIALS AND METHODS

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Plasmid Construction and Site-directed Mutagenesis-- An NdeI restriction site was created at the first ATG codon of the T. thermophilus aspC gene encoding AspAT in the plasmid pTG3 (8). The 2.0-kilobase pair DNA fragment of NdeI-EcoRI, which contains the entire T. thermophilus aspC gene, was ligated to the plasmid pET3a (Novagen). The resulting plasmid, pAMA, was used to overproduce T. thermophilus AspAT.

Mutations of the T. thermophilus aspC gene were produced by polymerase chain reaction (PCR)-available site-directed mutagenesis, using the mixed primer with the sequence 5'-GAGGAGACCATCGTCACCGTGGGGGGG(G/A/T/C)(G/A/T/C)(G/C)CAAGCGCTCTTCAACCTCTTCCAGG-3' and the primer with the sequence of 5'-GGTGGACTGGCTGGAGACGGAGGCCATGGC-3' to construct the K109V and K109S mutants. The plasmid carrying the T. thermophilus aspC gene was denatured at 98 °C for 5 min in the PCR mixture without DNA polymerase, Taq DNA polymerase was added, and 30 PCR cycles of 1 min at 98 °C, 1 min at 60 °C, and 2 min at 72 °C were performed. Each amplified fragment was subcloned into the pUC119 vector, and its DNA sequence was analyzed. No mutation was observed except for the designed mutation sites. The resultant DNA fragment carrying the mutant T. thermophilus aspC gene was cloned using PshAI and NcoI restriction sites into the pET3a expression vector, as described above for the wild-type aspC gene.

Amino Acid Sequence Alignment of Subgroup Ib AspATs-- We searched for proteins with amino acid sequences similar to that of T. thermophilus AspAT in the non-redundant protein sequence data base using the BLASTP program, version 2.0.4 (31), and alignment was carried out using the CLUSTAL W program, version 1.7 (32).

Enzyme Preparation-- The E. coli strain BL21(DE3) harboring the plasmid pLysE was transformed with pAMA or a plasmid carrying a mutant aspC gene. The transformants were cultivated at 37 °C in LB medium containing 50 µg/ml ampicillin and 20 µg/ml chloramphenicol; when the culture density reached 2 × 10⁸ cells/ml, isopropyl-1-thio--D-galactopyranoside was added to the medium to produce a concentration of 50 µg/ml. After incubation for a further 5 h, the cells were harvested by centrifugation and stored at 30 °C. T. thermophilus AspAT and the mutant enzymes were purified as described previously (8), and the N-terminal amino acid sequence of each purified enzyme was confirmed using an automated protein sequencer (ABI, model 473A). The concentrations of the PLP and PMP forms of T. thermophilus AspAT and its mutant enzymes were determined spectrophotometrically at 280 nm and pH 8.0 using molar extinction coefficients (_M) of 3.55 × 10⁴ and 3.45 × 10⁴ cm¹ M¹, respectively (5).

Kinetic Analysis-- A series of substrates used was identical to those described previously (29). The overall transamination activity was measured using a coupled assay method with malate dehydrogenase and NADH (33). The half-transamination reactions of the PLP and PMP forms of the enzymes were followed at 380 and 330 nm, respectively, using a stopped-flow spectrophotometer (Applied Photophysics, SX-17MV) as described previously (5). All the reaction conditions used were 50 mM HEPES, 100 mM KCl, pH 8.0, and 25 °C.

The kinetic parameters were determined using the following model and Equation 2,

(Eq. 2)

where k_app is the apparent rate constant at a given substrate concentration, k_max is the maximum rate constant, and K_d is the dissociation constant.

For slow kinetic experiments, a HITACHI U-3000 spectrophotometer was employed. When the k_app value was directly proportional to the substrate concentration, Equation 3, instead of Equation 2, was used to determine the catalytic efficiency, k_max/K_d (2).

(Eq. 3)

Binding of Inhibitors-- The aspartate analogs used were maleate and 2-methyl-DL-aspartate (2MeAsp). The maleate can bind noncovalently to the PLP form of AspAT, which increases the pK_a of the internal aldimine between N of Lys-258 and C4' of PLP (34, 35), whereas 2MeAsp binds to the PLP form of AspAT and forms an external aldimine complex. The K_d values of these inhibitors were obtained by fitting the absorbance changes at 430 nm (36) to theoretical curves.

Classification of the Modes of Interaction between Carboxyl Group Ligands and Protein Side Chains-- The Protein Data Bank (PDB) was searched to find structures complexed with ligands bearing carboxyl groups. If the carboxyl group of a ligand found in the PDB file was not a component of the natural ligand for the protein, we excluded the mode of interaction between such a carboxyl group and protein from our analysis. For example, the structure of fumarase C, which utilizes dicarboxylate, complexed with citrate (tricarboxylate) has been solved, and two of the three carboxylate groups of citrate were proposed to be significant (37). Therefore, the two significant carboxylate groups were included in, but the other was excluded from, our analysis. Some PDB proteins contain only part of the functional unit, in which case the additional symmetry-related atoms were generated or found in its original reference to analyze the intersubunit interactions. The interactions between ligands and proteins have been analyzed using the HBPLUS program (38) of McDonald and Thornton (39).

The mutual homologies of the PDB proteins used in this study were evaluated using the FASTA program (40). The amino acid sequences of the PDB proteins rarely agreed with the expected values estimated by the FASTA program, and the homology values for this data set were less than 0.05, which meant that almost all entries of the proteins selected for this study were not homologous with each other. However, three pairs of related proteins ((i) muscular and intestinal fatty acid binding proteins, (ii) dipeptide and oligopeptide binding proteins, and (iii) class  and  glutathione S-transferases) were included in our analyses because they have different ligand interaction modes. The class µ and  glutathione S-transferases were excluded because their interaction modes were similar to those of class  glutathione S-transferase.

	RESULTS

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Spectrophotometric Properties-- The mutant K109V and K109S T. thermophilus AspATs were constructed by replacing Lys-109 with Val and Ser, respectively, using PCR-based mutagenesis techniques. The circular dichroism (CD) spectra of the wild-type enzyme and mutant enzymes were almost identical in the far UV region (data not shown). Replacing of Lys-109 with Val increased the pK_a value of the Schiff base formed between the amino group of Lys-258 and C4' of PLP from 6.1 (8) to 6.5. Similarly, the K109S mutation increased the pK_a from 6.1 to 6.6. Such pK_a shift has been reported by the replacement of the Arg residue in the substrate-binding site of subgroup Ia AspAT with another residue (41- 44), and replacement of Arg-292 with a hydrophobic residue, Val or Leu, increased the pK_a value by 0.2 units (43). These results suggest the positive charge of Lys-109 was eliminated successfully from the active site of T. thermophilus AspAT without causing gross conformational changes. The increase of the pK_a value of the Schiff base of 0.4-0.5 resulting from Lys-109 mutation of T. thermophilus AspAT was somewhat larger than those (0.2 units) observed with the Arg-292 mutants of E. coli AspAT. This greater effect of the positively charged residue in T. thermophilus AspAT will be correlated to that the Lys-109 side chain is located in the substrate-binding pocket even when no substrate is present (Fig. 1).

An interesting observation was that the PMP form of T. thermophilus AspAT showed negative CD spectra in the 300-350-nm region (Fig. 3), which was certainly the signal from enzyme-bound PMP. The spectra of the two mutant enzymes were essentially identical to that of wild-type AspAT (data not shown). Such negative CD spectral peaks have not, as far as we are aware, been reported for AspATs. Both the PLP and PMP forms of E. coli AspAT and aromatic amino acid aminotransferase showed positive CD peaks in the 300-500-nm region (45, 46), and the PLP forms of AspAT (47) and ornithine aminotransferase (48) from the thermophile, Bacillus sp. YM-2 also showed positive peaks. Only the PLP forms of branched chain amino acid and D-amino acid aminotransferases, which are very distantly related to the other aminotransferases, showed negative CD peaks (49, 50). However, the PLP and PMP forms of T. thermophilus AspAT exhibited positive and negative CD peaks, respectively, and these spectrophotometric properties suggest that the electrostatic environment changed during the half-transamination reaction. These properties may be common to the subgroup Ib AspATs.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Circular dichroism spectra of wild-type AspAT. PLP form (solid line) and PMP form (dotted line) are shown. The ordinate shows molar ellipticity ([]M). Conditions: 50 mM HEPES, 100 mM KCl, 10 µM enzyme, pH 8.0, and 25 °C

Catalytic Properties-- The catalytic activities of the K109V and K109S mutant enzymes were about 10³ to 10⁴-fold lower than those of wild-type T. thermophilus AspAT under substrate concentrations of 10 mM aspartate and 20 mM 2-oxoglutarate (Table I). The R292V mutant enzyme of E. coli AspAT, which may correspond to the K109V mutant of T. thermophilus AspAT, showed catalytic activity 10⁵-fold less than that of the E. coli wild-type enzyme (43). Unfortunately, the k_cat and K_m values could not be determined due to the large K_m value of the acidic substrate for T. thermophilus AspAT mutants.

Pre-steady state kinetic studies were performed to evaluate further the energetic contribution of the Lys-109 side chain. The kinetic parameters for wild-type and two mutant enzymes are shown in Table II. The free energy differences (G_T) between the unbound enzyme plus substrate (E+S) and transition state (ES) for various substrates were calculated using the k_max/K_d values and the following equation (5, 51),

(Eq. 4)

where R is the gas constant, T the absolute temperature, k_B the Boltzmann constant, and h the Planck constant. The K109V mutant had larger G_T values (lower activities) when reacted with acidic substrates than the wild-type enzyme by 6-9 kcal·mol¹ ( G_T). Similar results were obtained for the K109S mutant, although its G_T values were slightly smaller than those of the K109V mutant. Conversely, when alanine was the substrate, the K109V and K109S mutants had smaller G_T values (higher activities) than the wild-type enzyme by 2-3 kcal·mol¹. These results indicate that both mutant enzymes retained the catalytic activity of the parent enzyme and had lost only the specific activity toward acidic substrates. The kinetic studies showed that the k_app value was directly proportional to the amino acid substrate concentration. Therefore, the k_max and K_d values for the reactions with acidic substrates could not be determined separately.

The affinities (1/K_d) of acidic substrate analogs (maleate and 2MeAsp) for the mutant and wild-type enzymes were evaluated spectrophotometrically. The wild-type T. thermophilus AspAT showed spectral changes when it was incubated with maleate (data not shown) and 2MeAsp, indicating that it bound to these analogs (Fig. 4), and the dissociation constants of the maleate and 2MeAsp were 11 mM at pH 6.5 and 15 mM at pH 8.0, respectively. However, the K109V and K109S mutant enzymes showed no spectral changes, not even in the presence of 20 mM 2MeAsp, indicating that the affinities of 2MeAsp for these mutant enzymes were very low (K_d > 500 mM). Furthermore, the apparent activities of the mutant enzymes were not altered by the presence of 2MeAsp, which kinetically suggested that 2MeAsp did not bind to these mutant enzymes.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Absorption spectra of wild-type AspAT (a) and its mutant enzymes (b) in the presence of 2-methyl-DL-aspartate. The values shown in the figure are the concentrations of 2-methyl-L-aspartate, which are half that of the racemic compound, 2-methyl-DL-aspartate.

Activity of T. thermophilus AspAT Toward Neutral Amino Acids-- T. thermophilus and E. coli AspATs show activity toward not only acidic but also hydrophobic substrates. Therefore, the natural amino acid substrates and a series of aliphatic substrates with different side chain lengths were used to elucidate the properties (volume, hydrophobicity, and steric hindrance) of the substrate-binding pockets of these enzymes (Table III). Consistent with the results of previous studies (28-30), the activity of T. thermophilus AspAT toward hydrophobic substrates increased as the side chain length of the aliphatic substrates increased (Fig. 5). An interesting finding was that neither T. thermophilus nor E. coli AspAT distinguished straight and branched substrates with seven or more carbons (29).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Correlation between GT and the carbon number of the aliphatic substrate. , T. thermophilus AspAT and substrates with straight side chains; , E. coli AspAT and substrates with straight side chains; , T. thermophilus AspAT and substrates with branched-terminal side chains; and , E. coli AspAT and substrates with branched-terminal side chains. GT is the free energy difference between the unbound enzyme plus substrate (E+S) and the transition state (ES) in the half-transamination reaction. Measurement conditions: 50 mM HEPES, 100 mM KCl, 10 µM enzyme, pH 8.0, 25 °C, and various concentrations of substrate.

T. thermophilus AspAT showed a stronger dependence on the substrate hydrophobicity than E. coli AspAT with 4- to 7-carbon substrates. The slope of the straight line fitted to the data for these substrates was 0.96 kcal·mol¹·CH₂¹ for T. thermophilus AspAT, which was considerably steeper than that for E. coli AspAT (0.73 kcal·mol¹·CH₂¹) (29). Therefore, T. thermophilus and E. coli AspATs showed similar activities toward 7-carbon substrate, although the activities of T. thermophilus AspAT toward alanine and 2-aminobutyric acid were lower (higher G_T values) than those of E. coli AspAT. The G_T values for the reactions of T. thermophilus AspAT with 7-, 8-, and 9-carbon substrates were virtually identical, whereas E. coli AspAT showed lower activities toward 8- and 9-carbon substrates than toward the 7-carbon substrate. These results suggest that T. thermophilus is subject to less steric hindrance and has a more flexible conformation than E. coli AspAT.

We also investigated the activities of T. thermophilus AspAT toward natural amino acid substrates and plotted the G_T values against the accessible surface areas of the amino acid side chains (5, 30, 45, 46, 52) (Fig. 6). The natural amino acid substrates used were divided into four groups: I, neutral substrates with short and medium-length side chains; II, positively charged substrates; III, -branched substrates; and IV, aromatic substrates, including histidine. T. thermophilus AspAT showed slightly lower activities toward class I substrates than E. coli AspAT (5), and both enzymes showed extremely low activities toward class II and III substrates, which is a property common to the group I aminotransferases. The characteristic difference between these two AspATs was in their activities toward class IV substrates. T. thermophilus AspAT showed no catalytic activity, whereas E. coli AspAT showed relatively high activity, toward aromatic substrates. An important finding was that the activities of the two AspATs toward methionine substrate were roughly similar, whereas those toward histidine, which has a similar accessible surface area to methionine, were quite different. The lower activity of T. thermophilus AspAT toward aromatic substrates may have been due to steric hindrance to aromatic side chains in the substrate-binding pocket or aromatic substrates binding to the pocket in nonproductive conformations. The binding of the apoenzyme of T. thermophilus AspAT to N-(5-phosphopyridoxyl)-L-phenylalanine suggested that the latter explanation was the most likely because binding of this ligand stabilized the enzyme considerably.³

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Correlation between GT and the accessible surface area of the amino acid substrate. GT is the free energy difference between the unbound enzyme plus substrate (E+S) and the transition state (ES) in the half-transamination reaction. The horizontal axis is the accessible surface area of the amino acid side chain (52). The natural amino acid substrates were classified into four groups: I, neutral substrates with small and medium side chains (black); II, positively charged substrates (blue); III, -branched substrates (green); IV, aromatic substrates including histidine (yellow). a, T. thermophilus AspAT. b, E. coli AspAT.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Structural Aspects of T. thermophilus AspAT-- The x-ray crystallographic structure of T. thermophilus AspAT (see Ref. 27; a more detailed description will be published elsewhere) revealed that the backbone conformation, or motif, was essentially similar to those of the subgroup Ia AspATs determined previously (19-26), even though their amino acid sequence identities were as low as 15%. This result is an example of the situation when proteins with dissimilar sequences show similar structural folding, and will be related to that the number of protein folds may be limited to 1000 (53) or 8000 (54). However, the three-dimensional structure of this enzyme yielded some information useful to the understanding of the molecular mechanism responsible for its stability, activity, and substrate specificity. T. thermophilus AspAT is highly stable under a wide range of temperature and pH conditions because the CD intensity of the PLP form of this enzyme at 222 nm did not change at temperatures up to 80 °C (8) or at pH values of 4 to 11.³ However, the native molecular mass of this enzyme was estimated to be 65 kDa by size-exclusion column chromatography (data not shown), much lower than that of the dimer structure (84 kDa) expected from other experiments and by analogy with the other homologs. These results suggest that this enzyme has a rigid conformation and highly packed structure. The B-factor values of its C atoms, which were derived from the crystallographic data, were relatively low for the whole structure (Fig. 7), indicating that this enzyme has a rigid (low degree of fluctuation) conformation. One exception to the low B-factor values was the N-terminal helix (16th-28th residues in the sequence of T. thermophilus AspAT), which showed substantially higher B-factor values than the average value for the whole structure (Fig. 7). This N-terminal helix is near the active site and a part of the substrate-binding pocket. Therefore, the high activities of this enzyme toward acidic substrates and broad neutral substrate specificity at room temperature (25 °C) may correlate with the flexibility of its N-terminal portion because the k_cat value (120 s¹) of wild-type T. thermophilus AspAT is as high as 50% of the k_cat value of E. coli AspAT (220 s¹) (5). It has been suggested that flexibility around the active site is required for enzymatic activity (55, 56). Conformational flexibility must reduce the steric hindrance in the substrate-binding pocket (29). Consequently, T. thermophilus AspAT has a thermostable rigid body and a flexible active site, which may be an ingenious strategy for an organism to adapt itself to environments with wide temperature ranges because T. thermophilus can grow at temperatures up to 85 °C (13). Therefore, we expect this enzyme to be a good model for investigating the relationships between conformational stability and enzymatic function and for engineering highly active and stable enzymes.

View larger version (41K): [in this window] [in a new window]   Fig. 7.   Backbone structure of aspartate aminotransferase. The color scale represents the B-factor value of the C atom. a, T. thermophilus AspAT in the substrate-free form (27). b, E. coli AspAT in the substrate-free form (22).

Role of the Lys-109 Residue-- In the substrate-free form of T. thermophilus AspAT, the -amino group of the Lys-109 side chain is located near the position equivalent to that of Arg-292 in the structure of subgroup Ia AspAT complexed with an acidic substrate (Fig. 1). This suggests that Lys-109 can interact with a distal COO of a substrate without changing its -amino group position. If so, the Arg side chain is probably longer than the optimal length for interactions, which may explain why Lys is conserved in subgroup Ib AspATs (Fig. 2).

The K109V and K109S mutant enzymes of T. thermophilus AspAT showed virtually no activity toward acidic substrates, whereas they were considerably more active toward neutral substrates than the wild-type T. thermophilus AspAT. The energetic contribution of the Lys-109 side chain to the enzymatic activity toward acidic substrates is about 6 kcal·mol¹ (Table II), very similar to the energy changes observed when the Arg-292 residue of E. coli AspAT was replaced by other residues (43). Consequently, Lys-109 residue of subgroup Ib AspAT is functionally compatible with Arg-292 of subgroup Ia AspAT. Lys-109 recognizes the distal carboxyl group of an acidic substrate and contributes to the high acidic substrate specificity of subgroup Ib AspATs.

The Modes of Interaction between Ligand Carboxyl Groups and Proteins-- Many three-dimensional structures of proteins and their complexes with ligands have been reported and collected by the PDB. The enzyme-substrate complex structures with ligand-bearing carboxyl groups were examined to show the preferred mode of interaction between each ligand carboxyl group and protein residue. We examined 60 carboxyl groups in 41 proteins (Table IV), which were not homologous with each other except for two fatty acid binding proteins (PDB codes 1HMS and 1ICM, 32% identity), two glutathione S-transferases (PDB codes 1GSE and 1PGT, 30% identity), and two peptide-binding proteins (PDB codes 1DPP and 2RKM, 24% identity). These related proteins were included in our analysis because the partners had different modes of interaction with carboxyl groups from the other. In 49 of the 60 cases, the Arg residue participated in the carboxyl group interaction (Table V), whereas the Lys side chain rarely (in only four cases) interacted with the ligand carboxyl group. His and Asn residue side chains interacted with the carboxyl group through their N atoms more often than Lys, and the use of the O atoms of Ser, Thr, and Tyr side chains was also observed frequently. An explanation for the preferences for Ser and Thr residues is that these residues can interact with the carboxyl group through two atoms of the same residue (the O atom of the side chain and N atom of backbone). Such a two-bonded interaction is topologically similar to the interaction mode of the Arg residue and occurred in eight of the structures we analyzed (PDB codes 1FDZ, 1GMS, 1GSE, 1LAF, 1PGT, 1YVE, 4ECA, and 7ACN). The frequency of interaction between the N atom of the peptide backbone and ligand carboxyl group was high frequency (30 cases), but the backbone O atom could not interact because of its lower potentiality as hydrogen donor (38).

When the modes of interaction between the Arg side chain and carboxyl group were classified into seven types (Scheme I), the "Ra" type, which is the recognition mode of the subgroup Ia AspATs, was the most frequent (the "Rf" type was ignored because its type takes various conformation). This preponderance of the Ra type may mean that this is the strongest interaction of the seven types, and it does not need additional assistant residues for complete charge compensation. Therefore, the Ra type interaction may occur readily during the process of protein evolution.

View larger version (12K): [in this window] [in a new window]   Scheme I.

The modes of interaction between positively and negatively charged residues have been investigated thoroughly (57, 58), and the "Rc" type occurred the most frequently, followed by the Ra type, among 484 Arg-Asp/Glu residue pairs (57). Therefore, the Ra type of interaction between the Arg side chain and carboxyl group may be characteristic of the interaction between a ligand carboxyl group and a protein residue.

Carboxyl Group Recognition by AspATs-- Site-directed mutagenesis and x-ray crystallographic studies (27) on T. thermophilus AspAT indicated that the Lys-109 residue recognized the distal carboxyl groups of dicarboxylate substrates, whereas the Arg-292 residue in subgroup Ia AspATs performed this function. However, both subgroup Ia and Ib AspATs utilize the side chain of Arg-386 to recognize the -COO of a substrate, and their interaction modes are the Ra type. Our search for the modes of recognition of the ligand carboxyl groups utilized by other enzymes revealed that the Arg residue was preferred for this purpose, and recognition by Lys was extremely rare. This preference for Arg was not due to the distribution of amino acid residues, because both Arg and Lys are coded with almost equal frequency in the E. coli K-12 strain MG1655 (59) and cyanobacterium Synechocystis sp. strain PCC6803 (60) genomes (Table VI). For example, the Arg and Lys residue coding frequencies in the E. coli genome are 5.5 and 4.4%, respectively (59). Lys is preferred to a greater extent in Bacillus subtilis (the Arg and Lys coding frequencies are 4.1 and 7.1%, respectively) (61). In the case of T. thermophilus, the Arg and Lys coding frequencies are 7.9 and 4.9%, respectively. However, x-ray crystallographic (27) and site-directed mutagenesis studies revealed that T. thermophilus AspAT utilized the Lys residue (Lys-109) as the determinant for acidic substrate specificity. This is expected to be common to the subgroup Ib AspATs because this Lys residue is well conserved among these enzymes.

X-ray crystallographic studies on the structures of T. thermophilus AspAT complexed with dicarboxylic substrate analogs and the mutant enzymes described above are in progress (the details will be reported elsewhere), and we expect these structures will reveal the precise topologies of the interactions between protein residues and substrates. Residues other than Lys-109 may work as "assistants" to fix the orientation of a substrate's carboxyl group.

Finally, some subgroup Ib aminotransferases do not have a Lys residue at their 109th position. Although their enzymatic activities have not been characterized, they may turn out not to be "aspartate" aminotransferases, but aminotransferases with different substrate specificities.

	FOOTNOTES

^* This work was supported in part by Grants-in-Aid for Scientific Research 09680619 and 07558224 from the Ministry of Education, Science, Sports, and Culture of Japan and "Research for the Future" Program Grant JSPS-RFTF96L00506 from the Japan Society for the Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 81-6-850-5433; Fax: 81-6-850-5442; E-mail: kuramitu{at}bio.sci.osaka-u.ac.jp.

The abbreviations used are: PLP, pyridoxal 5'-phosphate; AspAT, aspartate aminotransferase; 2MeAsp, 2-methyl-DL-aspartate; PCR, polymerase chain reaction; PDB, Protein Data Bank; PMP, pyridoxamine 5'-phosphate.

² The amino acid residues are numbered according to the sequence of pig cytosolic aspartate aminotransferase (62).

³ Y. Nobe, S.-I. Kawaguchi, H. Ura, T. Nakai, K. Hirotsu, R. Kato, and S. Kuramitsu, unpublished results.

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