Homologous Alkalophilic and Acidophilic l-Arabinose Isomerases Reveal Region-Specific Contributions to the pH Dependence of Activity and Stability

Abstract

To study the pH dependence of l-arabinose isomerase (AI) activity and stability, we compared homologous AIs with their chimeras. This study demonstrated that an ionizable amino acid near the catalytic site determines the optimal pH (pH_opt) for activity, whereas the N-terminal surface R residues play an important role in determining the pH_opt for stability.

TEXT

Unlike neutrophilic enzymes, many proteins expressed by acidophiles and alkalophiles exhibit maximal activities beyond neutral pH conditions (3, 5, 12, 19, 25). It has been noted that the optimum pH (pH_opt) of the maximal catalytic rate of an enzyme is primarily affected by the pK_a values of ionizable groups on the catalytic and substrate binding sites (1, 19, 28, 29). On the other hand, the pH_opt for maximal protein stability, defined as the pH at which the free energy of unfolding (ΔG_U) is lowest, is likely to be dependent on the base/acid ratio of surface groups (27) and the net charge of each specific structural region (6, 24). A numerical approach (1) demonstrated that pH stability is not directly related to the isoelectric point (pI) of a protein (4). Denaturation experiments also suggested that the pH dependence of the thermodynamic stability (ΔG_U) of a protein arises as a consequence of differential pK_a values attained in the folded and unfolded states (2, 27).

Thermostable l-arabinose isomerases (AI; EC 5.3.1.4) are utilized for the production of d-tagatose as a sugar replacement (13, 15, 18, 22). To achieve high yields of d-tagatose production, isomerization at high temperatures (∼60°C) and acidic pH (≤6.0) is considered the most favorable condition for minimizing unwanted byproduct formation and browning effects (8, 19). Hence, for the rational design and engineering of a thermoacidophilic AI, the relationships between sequence, structure, and function with respect to the pH dependence of AIs must be better understood. Therefore, we characterized the thermoalkalophilic AI from a newly isolated Alicyclobacillus sp. TP7 strain in comparison with its acidic counterpart (19). Although the two AIs showed >82% amino acid sequence similarity, their pH_opt values differ by 3 to 3.5 U. To better understand the molecular basis of differences in the pH dependence of their activity and stability, we generated AI chimeras by homologous recombination and site-directed mutagenesis and characterized them.

Isolation of the araA gene and characterization of AI.

To isolate thermoacidophiles producing AIs, over 200 samples from hot springs in Indonesia were cultured on EM-1 medium (16) supplemented with l-arabinose at pH 2 and 65°C (see the supplemental material). From these, 20 strains were isolated and assayed for AI activity as described in reference 10. Isolate TP7, which showed the highest AI activity, was selected and its 16S rRNA gene sequence was determined (30). Sequencing revealed a 97% to 99% similarity with Alicyclobacillus strains (see Fig. S1 in the supplemental material). Therefore, isolate TP7 was identified as Alicyclobacillus sp. TP7 (Korean Collection for Type Cultures 33088). To obtain the araA gene encoding AI, the degenerate primers DaraAF and DaraAR were designed based on the internal amino acid sequences of araA genes (see the supplemental material) (19). The intact araA gene of Alicyclobacillus sp. TP7 (GenBank accession number JQ945232) was amplified by PCR, and the resulting DNA was cloned into the pET 22b(+) vector to generate pET-ASAI. Sequence analysis demonstrated that the 1,485-bp araA gene encoded a 494-amino-acid polypeptide (Alicyclobacillus sp. TP7 AI [ASAI]). The presumed ASAI amino acid sequence showed high (68% to 96%) similarity to those of other AIs from A. acidocaldarius (7, 19, 21) and Geobacillus stearothermophilus (17).

The characterization of purified ASAI is summarized in Table S1 in the supplemental material. The relative molecular weight (M_r) of ASAI was estimated to be 56 based on SDS-PAGE. However, native PAGE and gel filtration chromatography suggested that the native form of ASAI exists as a homotetramer (M_r = ∼220). The pH_opt of ASAI activity was 8.5 at 70°C, and ASAI retained its original activity (>95%) even after 20 h of incubation at pH 7 to 9. The K_m and V_max of ASAI for l-arabinose were determined to be 49.7 mM and 52.4 μmol/mg · min at pH 8.5 (70°C). ASAI was activated by the addition of 1 mM Mn²⁺ (2.0-fold) and 1 mM Mg²⁺ (1.5-fold), whereas 1 mM EDTA inhibited AI activity (17–19).

Generation of functional AI chimeras.

The crystal structure of Escherichia coli l-arabinose isomerase (ECAI) (20), together with the results of our previous work (14, 19), suggested that the N- and C-terminal domains of AIs might contribute to the physicochemical properties of activity and stability, respectively. In addition, K269 in Alicyclobacillus acidocaldarius AI (AAAI) was found to play an important role in determining the pH_opt (19, 22). Based on these data, a comparison of the sequence of the alkaline ASAI with the sequences of acidic AAAI and neutral Geobacillus stearothermophilus AI (GSAI) revealed two remarkable features (see Fig. S2 in the supplemental material). First, ASAI contains E267, which corresponds to K269 in AAAI as the “A residue,” defined as ionizable groups that are directly involved in catalysis by amino acids with different charge or pK values (19, 26, 28). This was further confirmed using the hybrids I-E267K and II-K269E (see below). Second, ASAI was richer in R, which has the highest pK_a value, than AAAI (see Table S2 in the supplemental material). To investigate these characteristics, we generated several chimeric enzymes (see the supplemental material). In these chimeras, the N- or C-terminal regions were exchanged with the corresponding regions of their acidic or alkaline counterparts (Fig. 1A). The four chimeras were expressed as catalytically active and soluble forms in E. coli BL21(DE3) and purified for detailed characterization.

Fig 1.

Schematic diagrams of the wild-type AIs and their chimeras. (A) Primary structures of the wild-type AIs and chimeras (see the supplemental material). The blue bar represents the araA gene encoding Alicyclobacillus sp. TP7 AI (ASAI), and the green bar indicates the araA gene encoding A. acidocaldarius AI (AAAI). (B) pH dependence of the wild-type AIs and chimeras based on their catalytic activity. The V_max data were plotted as a function of pH and fitted to a bell-shaped activity profile from the equation V_max = (V_max)_m × [1 / (1 + 10^−pH / 10^−pKa1 + 10^−pKa2 / 10^−pH)], where apparent pK_a values corresponding to the acidic (pK_a1) and basic (pK_a2) limbs were determined by nonlinear least-squares fitting. Sp act, specific activity. (C) The hypothetical three-dimensional (3D) structures of AAAI, GSAI, and ASAI based on the crystal structure of ECAI (Protein Data Bank [PDB] 2AJT) were generated with the SWISS-MODEL program, and the locations of distinct charged amino acids on their surface and side chains of amino acid residues are depicted in spheres. Carbon, nitrogen, oxygen, and sulfur atoms are indicated in white, blue, red, and yellow, respectively. GSAI, G. stearothermophilus AI.

pH dependence of the activity and stability of hybrid AIs.

For comparison with the wild-type AIs, analogous studies of the pH dependence of activity and stability were conducted with four hybrids. Hybrid I showed a pH_opt range of 7.5 to 8.0, whereas hybrid II exhibited a pH_opt range of 6.5 to 7.0, and its pH activity profile was similar to that of AAAI (Fig. 1B). Notably, the optimal temperatures (T_opt) of all hybrid AIs ranged from 60 to 70°C, indicating that they had retained their thermophilic properties (Table 1). To investigate whether the A residue can alter the pH_opt of hybrids I and II, the effect of pH on activity was studied using hybrids I-E267K and II-K269E. Hybrid I-E267K showed a pH_opt range of 7.0 to 7.5, although its pH stability was maximal at pH 9. Likewise, although the hybrid II-K269E pH_opt of 6.5 to 7.0 was altered to 7.5, its pH stability was maximal at pH 7.

Table 1.

pH dependence of the activity and stability of wild-type and hybrid AIs

Enzyme	TempReact (°C)a	pHopt	pH	pH1/2 (h)b
ASAI	70	8.5–9.0	5	6.9 ± 0.3
			7	201.0 ± 13.4
			9	334.0 ± 37.1
AAAI	65	6.0–6.5	5	14.0 ± 1.1
			7	90.0 ± 8.2
			9	64.6 ± 4.2
Hybrid I	65	7.5–8.0	5	10.5 ± 0.0
			7	86.0 ± 2.5
			9	94.1 ± 5.9
Hybrid II	60	6.5–7.0	5	14.8 ± 1.1
			7	59.0 ± 5.8
			9	43.0 ± 1.8
Hybrid I-E267K	65	7.0–7.5	5	8.5 ± 0.5
			7	71.7 ± 3.4
			9	97.1 ± 6.3
Hybrid II-K269E	60	7.5	5	14.8 ± 1.8
			7	88.5 ± 5.2
			9	70.0 ± 6.5

^aTemp_React, reaction temperature.

^bThe pH_1/2 is the half-life (t_1/2) of AI activity at various pH values.

To examine the pH dependence of protein stability in detail, the GdnHCl-induced unfolding of hybrid AIs at 25°C was investigated by monitoring the circular dichroism (CD) signals (see Fig. S3 in the supplemental material). Unfolding of the AIs involves highly cooperative transitions between the native and unfolded states, with no detectable intermediates. The midpoint concentration (C_m) of the unfolding transition for ASAI at pH 9 (2.4 M) was higher than that of AAAI (1.6 M). On the other hand, the C_m value for ASAI at pH 5 (0.8 M) was lower than that of AAAI (1.2 M). Hybrid I and hybrid II showed similar C_m values that were higher at pH 9 than at pH 7. The transitions of hybrids I and II at pH 5 were 0.5 M for hybrid I and 1.0 M for hybrid II, whereas their C_m values at pH 9 were 1.9 M and 1.6 M, respectively. These pH stability patterns coincided with those derived from the wild-type AIs, indicating that the pH stability of AI is more dependent on the N-terminal domain. Therefore, these data demonstrate that the pH_opt for activity and stability of AI can be altered independently.

Characterization of AI single mutants.

Based on the idea that the highly conserved residues may not be responsible for the pH dependence of AIs, the amino acid sequences were aligned to identify residues in acidic and alkaline AIs not present in neutral AIs (see Fig. S2 in the supplemental material). The roles of those residues in the pH dependence of AI were then investigated. Amino acids were selected based on the following criteria: (i) titratable charged amino acids were considered; (ii) amino acids that were also found in neutrophilic AIs were excluded; (iii) amino acids in ASAI that had charge properties opposite those of corresponding residues in AAAI were included; and (iv) amino acids in the N-terminal domain of AIs were considered. Based on the above criteria, 12 R residues in ASAI were selected (Fig. 1C) and replaced with the corresponding residues from AAAI and vice versa (Table 2). Remarkably, ASAI mutants had the pH_opt of stability shifted downward from basic to acidic pH, whereas the pH_opt values for the stability of AAAI mutants were shifted upward from acidic to basic pH. Nevertheless, all single mutants showed T_opt values similar to those of the wild-type enzymes, indicating that substitution in these residues affected neither the T_opt nor the pH_opt for activity. Therefore, these data strongly suggest that these R residues in the N-terminal domain of ASAI appear to be responsible for the stability at alkaline pH.

Table 2.

pH stability of the wild-type AIs and their single-amino-acid mutants

Enzyme	TempReact (°C)a	pHopt	pH	pH1/2 (h)b
ASAI	70	8.5	5	6.9 ± 0.3
			7	201.0 ± 13.4
			9	334.0 ± 37.1
AAAI	65	6.0–6.5	5	14 ± 1.1
			7	90.0 ± 8.2
			9	64.6 ± 4.2
ASAI-R31E	70	6.0–8.0	5	10.1 ± 0.2
			7	125.2 ± 10.4
			9	250.9 ± 20.9
AAAI-M33R	70	7.0–8.0	5	9.1 ± 0.3
			7	124.1 ± 10.3
			9	142.0 ± 20.3
ASAI-R48E	70	6.0–8.0	5	8.9 ± 0.3
			7	192.9 ± 12.4
			9	273.7 ± 24.9
AAAI-V50R	65	7.0–8.0	5	7.8 ± 0.3
			7	158.4 ± 8.3
			9	231.6 ± 17.8
ASAI-R200E	65	6.0–8.0	5	9.8 ± 0.1
			7	100.3 ± 10.0
			9	250.9 ± 20.9
AAAI-Q202R	70	7.0–8.0	5	11.8 ± 0.2
			7	100.3 ± 3.3
			9	83.2 ± 4.6
ASAI-R216E	70	7.0–7.5	5	8.6 ± 0.3
			7	200.7 ± 13.4
			9	301.0 ± 30.1
AAAI-Y218R	70	7.0–7.5	5	10.4 ± 0.1
			7	124.6 ± 5.2
			9	102.8 ± 10.6

^aTemp_React, reaction temperature.

^bThe pH_1/2 is the half-life (t_1/2) of AI activity at various pH values.

Overall, these empirical data determined with AIs were consistent with Alexov's numerical calculations (1) indicating that the pH_opt of activity is not correlated with the pH_opt of stability (11). Therefore, this report suggests that such region-specific charged amino acids are likely to have evolved to adapt the stability of enzymes at a specific pH in a manner independent of catalytic activity. Further, altering the charged state of amino acids near catalytic sites (9, 19, 23, 28, 31) and the net charge of a region of the protein might be an efficient way to engineer the pH dependence of the activity and stability of enzymes (1, 24, 27, 28).

Nucleotide sequence accession number.

The 16S rRNA gene sequence of isolate TP7 was submitted to GenBank under accession number JX218020.