Abstract
Catalase has crucial role in adaptive response to H2O2. Main channel structure responsible for substrate selectivity was estimated to understand the relationship between the evolutionary direction of catalases from Exiguobacterium oxidotolerans and Psychrobacter piscatorii which survive in cold and high concentration of hydrogen peroxide, and their catalytic property. E. oxidotolerans catalase (EKTA) exhibited a higher ratio of compound I formation rate using peracetic acid (a substrate lager than H2O2)/catalase activity using H2O2 as the substrate than P. piscatori catalase (PKTA). It was considered that the ratio was attributed to the size of the amino acid residues locating at the bottle neck structure in the main channel. The differences in the ratio of the compound I formation rate with peracetic acid to catalase activity with H2O2 between the deeper branches in the phylogenetic tree in both EKTA and PKTA were large. This indicates that catalases from the hydrogen peroxide-tolerant bacteria have evolved in different directions, exhibiting effective catalytic activity and allowing broader substrates size or H2O2-specific substrate acceptability in EKTA and PKTA, respectively. It is considered that the main channel structure reflected the difference in the evolutionary direction of clade 1 and clade 3 catalases.
Keywords: Bottleneck, Heme catalase, Main channel, Size of amino acids
Introduction
Bacterial monofunctional catalases are classified into clades 1–3 according to phylogenetic analysis based on their amino acid sequences [1–5]. Clade 1 catalases contain approximately 500 amino acid residues per subunit and are mainly of plant origin, except a subgroup that is of bacterial origin. Clade 2 catalases, which exhibit larger molecular mass than the catalases from other clades, consist of approximately 750 amino acid residues. The catalases in this clade originated from fungi, bacteria and archaea. Clade 3 catalases, with nearly 500 residues per subunit, occur in archaea, bacteria, fungi and some other eukaryotes. We purified the clade 1 and 3 catalases, Exiguobacterium oxidotolerans (EKTA) and Psychrobacter piscatorii catalase (PKTA), respectively, from bacteria isolated from the upstream wastewater in a food processing factory that used H2O2 [6, 7].
Genera Exiguobacterium and Psychrobacter include several cold-adapted species [8, 9]. They often colonize the same ecological niches such as Siberian permafrosts and Antarctic sea ice probably due to their ability to withstand freezing or freeze-thawing stress. Considering the isolation of genera Exiguobacterium and Psychrobacter from cold regions and high concentration of H2O2 containing environments, there may be similar molecular mechanisms that help adapt to both freeze-thawing environments and H2O2 containing environments.
It has been shown that main channel structure in the catalase plays a crucial role in the catalysis [10–12]. Comparison of the structural and functional data on EKTA a clade 1 catalase, with those of two clade 3 catalases (bovine liver catalase [BLC] and Micrococcus luteus [MLC]) revealed that the size of the bottleneck defines the rate of compound I formation, which corresponds to the size of the substrate molecule. The atom-to-atom distance for combinations of amino acid residues showed that, the Leu149–Ile180 and Asp109–Met167 combinations at the bottleneck of EKTA lead to larger bottleneck size than the combination in BLC and MLC [10]. The sizes of the amino acids and the probability of occurrence of the corresponding amino acids (based on comparison of catalase sequences in the database) indicate that Met167 may play a key role in determining the size of the bottleneck of EKTA.
To understand the relationship between the evolutionary direction of Exiguobacterium oxidotolerans catalase (EKTA) and Psychrobacter piscatorii catalase (PKTA) affected by cold and hydrogen peroxide and their catalytic property, main channel structure concomitant with substrate selectivity was estimated. The results demonstrate the evolutional direction of catalases from genera Exiguobacteium and Psychrobacter which exhibit high adaptability for extreme environments is reflected by the evolutionary direction of the clade which each catalase involved.
Materials and Methods
Chemicals, Enzymes, and Resins
Standard chemicals were purchased from Wako Pure Chemicals (Osaka, Japan). The chemicals were of the highest grade available and were used without further purification. Peracetic acid (Sigma-Aldrich, St. Louis, MO, USA) was treated with a trace amount of catalase (~ 2 nM) in 50 mM potassium phosphate buffer (pH 7.0) to eliminate H2O2 before use [13]. The concentration of peracetic acid was determined based on an extinction coefficient (ϵ) of 2.62 × 104 M−1 cm−1 at 353 nm for triiodide generated via oxidation of potassium iodide [14]. BLC and MLC were purchased from Sigma-Aldrich and Nagase Chemtex, respectively. The anion exchange resin Q-Sepharose Fast Flow, hydrophobic chromatography resin Phenyl Sepharose High Performance and gel filtration resin Sephacryl S-300 were purchased from GE Healthcare (Uppsala, Sweden). The hydroxyapatite resin was purchased from Seikagaku Kogyo (Tokyo, Japan). The anion exchange resin DEAE-Toyopearl 650 M was purchased from Tosoh Corporation (Tokyo, Japan).
Bacterial Strains
Exiguobacterium oxidotolerans T-2-2T was cultured and its catalase was purified as described previously [10]. P. piscatorii T-3 was cultured, and its catalase was purified as described previously [15]. To obtain the catalase gene (KatA) from Deinococcus radiodurans NBRC 15346T was obtained from Biological Research Center, NITE (National Institute of Technology and Evaluation). D. radiodurans strain NBRC 15346T was cultivated aerobically at 30 °C in a medium containing 10 g polypeptone (Nihon Pharmaceutical, Tokyo, Japan) and 2 g yeast extract (Kyokuto, Kyokuto, Japan). Catalase-less Escherichia coli UM255 harboring the Pseudomonas syringae catalase (PSCF) gene in the pEC3E56 plasmid was a kind gift from Dr. PC Loewen (University of Manitoba, Canada) and was cultured as described previously [16].
Construction of Expression Vectors for Recombinant P. syringae Catalase (PSCF)
To construct a strong PSCF-expressing recombinant vector, the entire PSCF sequence in pEC3E56 was amplified using polymerase chain reaction (PCR) using the primers 1 (5′-CACCATGCCGTTATTAAACTGGTCCAGAC-3′) and 2 (5′-TCAGTCCTTGAGGCTTGCAG-3′), and the product was inserted into the TOPO cloning site of pET101/D-TOPO (Invitrogen). To construct a bottleneck amino acid-substituted mutant PSCF (F170L), PCR was performed using the mutagenic primers 3 (5′-CCGGACATGGTCCACGCGTTAAACCTGATC-3′) and 4 (5′-AACGCGTGGACCATGTCCGGGAACTTGATG-3′) with the constructed vector as the template. Then, the PCR product was ligated via PCR using primers 1 and 2 and inserted into the TOPO cloning site of pET101/D-TOPO. As E. coli UM2 does not contain T7-polymerase, which is required for expression from pET101/D-TOPO, pVLT49 [17], which harbors the T7-polymerase-encoding vector was also introduced into this expression system. The constructed vectors and pVLT49 were transformed into catalase-less E. coli UM255 via heat shock and transformation and storage solution (TSS) methods [18], respectively.
Construction of Expression Vectors for Recombinant D. radiodurans Catalase (KatA)
To construct a strong catalase-expressing recombinant vector, the entire KatA sequence of D. radiodurans NBRC 15346T was amplified. The first PCR was performed using the primers F2 (5′-AGAATGATTCTCAATATGGTGCAG-3′ [77–54 bp upstream of the catalase sequence]) and R2 (5′-TTCAGTTTCCGTGCTCAGC-3′ [4–24 bp downstream of the catalase sequence). The second PCR was performed using F1 (5′-CACCATGAGCGACGAAAACAACAAG-3′ [1–21 bp of the catalase sequence]) and R1 (5′-TCAGTACAGGCTGCTCGCCTC-3′ [1611–1591 bp of the catalase sequence]). Chromosomal DNA of D. radiodurans NBRC 15346T and the product of the first PCR were used as the template for first and second PCRs, respectively. The product was inserted into the TOPO cloning site of pET101/D-TOPO (Invitrogen). The constructed vectors and pVLT49 were transformed into catalase-less E. coli UM255 via heat shock and TSS methods [18], respectively.
Expression of Catalase from Recombinants
The catalases were purified from the E. coli UM255 recombinants constructed in this study using a 2-L baffled Erlenmeyer flask containing 1 L of 2 × Luria–Bertani (LB)-medium supplemented with 60 μg mL−1 ampicillin, 50 μg mL−1 streptomycin and 34.4 μg mL−1 chloramphenicol. After adding 150 mL of the seed culture, the flask was incubated at 27 °C at 80 rpm. When the culture reached optical density (OD600) of 0.5, 1 mM 5-aminolevulinic acid (ALA) and 0.1 mM iso-propyl-β-D-thiogalactopyranoside (IPTG) were added, and the cells were harvested after 24 h of cultivation. The cells were harvested via centrifugation at 7000 × g for 20 min at 4°C and stored at − 30°C.
Purification of Catalases
EKTA, MLC, and BLC were purified as describes previously [10]. PKTA was purified using the method of Kimoto et al. [15]. PSCF and D. radiodurans KatA were purified from recombinant E. coli UM255 harboring catalase genes in pET101/D-TOPO disrupting the cells using a French press. PSCF and its bottleneck amino acid substituted mutant PSCF (F170L) were purified using anion exchange chromatography, on Q-Sepharose Fast Flow, hydroxyapatite chromatography and hydrophobic chromatography on Phenyl Sepharose High Performance column (Tables 1, 2). D. radiodurans KatA was purified via anion exchange chromatography on DEAE-Toyopearl 650 M and gel filtration chromatography on Sephacryl S-300 (Table 3). Samples of all catalases used in this study were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to assess their purity. SDS-PAGE was performed as described previously [15].
Table 1.
Purification of PSCF from E. coli UM255 harboring PSCF coding pET101/D-TOPO and T7-polymerase coding pVLT49
| Step | Total protein (mg) | Total activity (U × 103) | Specific activity (U/mg) | Purification (fold) | Yield (%) |
|---|---|---|---|---|---|
| Crude extract | 1570 | 13,500 | 8620 | 1.0 | 100 |
| Q-sepharose Fast Flow | 349 | 10,100 | 28,900 | 3.4 | 75 |
| Hydroxyapatite | 64.6 | 6330 | 98,000 | 11.0 | 47 |
| Phenyl Sepharose High Performance | 13.8 | 2310 | 167,000 | 19.0 | 17 |
Table 2.
Purification of PSCF (F170L) from E. coli UM255 harboring PSCF coding pET101/D-TOPO and T7-polymerase coding pVLT49
| Step | Total protein (mg) | Total activity (U × 103) | Specific activity (U/mg) | Purification (fold) | Yield (%) |
|---|---|---|---|---|---|
| Crude extract | 1550 | 10,600 | 6800 | 1.0 | 100 |
| Q-sepharose Fast flow | 446 | 7990 | 17,900 | 2.6 | 75 |
| Hydroxyapatite | 60.8 | 3930 | 64,600 | 9.5 | 37 |
| Phenyl Sepharose High Performance | 15.1 | 1450 | 96,000 | 14.0 | 14 |
Table 3.
Purification of D. radiodurans KatA from E. coli UM255 harboring PSCF coding pET101/D-TOPO and T7-polymerase coding pVLT49
| Step | Total protein (mg) | Total activity (U × 103) | Specific activity (U/mg) | Purification (fold) | Yield (%) |
|---|---|---|---|---|---|
| Crude extract | 5670 | 10,530 | 1860 | 1.0 | 100 |
| DEAE-Toyopearl 650 M | 159 | 7250 | 46,000 | 25 | 69 |
| Sephacryl S-300 | 29.0 | 4600 | 159,000 | 85 | 44 |
Determination of Catalase Activity
Catalase activity was measured spectrophotometrically by monitoring the initial decrease in absorbance at 240 nm caused by the disappearance of H2O2 using a Cary 100 bio-spectrophotometer (Varian, Walnut Creek, CA) at 25°C. The concentration of H2O2 was determined on the basis of an extinction coefficient of 43.6 M−1 cm−1 [19]. The standard reaction mixture for the assay contained 50 mM potassium phosphate buffer (pH 7.0), 30 mM H2O2 and 10 μL of catalase solution in a total volume of 1.0 mL. The amount of enzyme activity that decomposed 1 μmol of H2O2 per min was defined as 1 U. The enzyme activity was expressed as the mean of at least four measurements.
Kinetic Measurements of the Formation of the Reaction Intermediate, Compound I, with Peracetic Acid
The concentration of peracetic acid was determined by measuring the absorbance at 353 nm, which was due to the formation of triiodide ions via oxidation of potassium iodine. Catalases were reacted with peracetic acid using equal volumes of several concentrations of peracetic acid and catalase (final concentration, 0.5 mM) dissolved in 50 mM sodium phosphate buffer (pH 7.0) at 5°C. The spectral changes in the Soret peak during compound I formation were recorded on a Unisiku RSP-1000 stopped-flow spectrophotometer (Osaka, Japan). The velocity constant (s−1) was estimated by measuring the reduction rate of the Soret peak absorbance and calculated using the least-square method. The kinetic constant (M−1 s−1) was determined by varying the peracetic acid concentration under pseudo-first-order reaction conditions.
Amino Acid Sequence Analysis in the Database
The probability of occurrence of amino acid residues in the bottleneck of the main channel of catalases was estimated using 415 catalase sequences that were equivalent to the seed sequences in the EMBL-EBI database (http://pfam.xfam.org/family/PF00199#tabview=tab3). Multiple alignments of the sequences were performed using the MUSCLE program [20]. A phylogenetic tree was constructed using the maximum-likelihood method based on the JTT matrix-based model [21] of MEGA X [22]. The evolutionary distance matrix was calculated using Kimura’s two-parameter model. The confidence values for the branches of the phylogenetic tree were determined using bootstrap analysis.
Results
Catalase Activity and Compound I Formation Rate with Peracetic Acid as the Substrate
The activities of the purified catalases obtained using 30 mM H2O2 and compound I formation rates using peracetic acid (a substrate lager than H2O2) are shown in Table 1. EKTA exhibited a higher ratio of compound I formation rate using peracetic acid/catalase activity using H2O2 as the substrate (b/a ratio in Table 4) than PKTA. As there is a possibility on the difference between EKTA (clade 1) and PKTA (clade 3) catalases in b/a ratio is attributed to the difference in the clade involved, catalase activity using 30 mM H2O2 and compound I formation rate with peracetic acid as the substrate were compared using several catalases involved in each clade (Table 4). Among the bacterial catalases, with the exception of EKTA, clade 3 catalases exhibited higher catalase activity for H2O2 than clade 1 catalases, whereas clade 3 catalases exhibited lower compound I formation rates with peracetic acid than clade 1 catalases. Comparison of the b/a ratios between clade 1 and clade 3 catalases showed that the values for clade 1 were higher than those for clade 3. Therefore, it is considered that the differences in EKTA (clade 1) and PKTA (clade 3) catalases due to the difference in the clade involved.
Table 4.
Comparison of the catalase activity in the presence of 30 mM H2O2 (a), compound I formation rate with peracetic acid (b), and the reaction ratio of the compound I formation rate with peracetic acid to catalase activity using 30 mM H2O2 (b [large substrate]/a [small substrate])
| A | b | |||
|---|---|---|---|---|
| Catalase | Catalase activity (× 102 U mg protein−1) | Compound I formation rate with peracetic acid (× 102 M−1 s−1) | b/a-ratio | Amino acid residue corresponding to W185 in BLC |
| Clade 1 | ||||
| EKTA | 4300 | 5950 | 1.4 | M (167.7)b |
| PSCF | 1670 | 1100 | 0.66 | F (193.5) |
| PSCF (F170L) | 960 | 872 | 0.91 | L (164.6) |
| D. radiodurans KatA | 1590 | 510 | 0.32 | F (193.5) |
| Clade 3 | ||||
| BLC | 1000 | 67.9a | 0.068 | W (231.7) |
| MLC | 3200 | 4.40a | 0.0014 | W (231.7) |
| PKTA | 2220 | 12.4 | 0.0056 | W (231.7) |
It was considered that the difference in b/a ratios between clade 1 and clade 3 attributed to the size of the amino acid residues strongly correlated with substrate selection. To understand the relationship between the amino acid residue corresponding to Trp185 in BLC with activity, which may define the size of the bottleneck, an amino acid-substituted mutant PSCF enzyme was produced. PSCF (F170L) exhibited lower catalase activity (9.6 × 104 U mg protein−1) than the original catalase (1.8 × 105 U mg protein−1). The F170L mutation in PSCF widened the bottleneck of the main channel of the catalase, as the volume of Leu is lower than that of Phe. However, the structural change concomitant with this mutation is probably not suitable for the reaction with H2O2. To understand the effect of the amino acid substitution, the ratio of the compound I formation rate to catalase activity using 30 mM H2O2 was estimated (b/a ratio in Table 4). Although PSCF (F170L) exhibited lower catalase activity than the original PSCF, it exhibited a higher b/a ratio. This suggested that the size of the amino acid residue corresponding to Trp185 in BLC defines the acceptability of larger substrates, such as peracetic acid, which contains a hydrophobic part.
Although we observed the difference between clade 1 and clade 3 catalases, the b/a ratio was varied within the same clade. This may be attributed to total structural differences in the main channel. The bottleneck amino acid residues consisted of two combinations of amino acid residues, namely Asp113 (BN1 in Table 2) and Trp185 (BN3), and Gln167 (BN2) and Lue198 (BN4), which corresponded to those in BLC (Figs. 1, 2; Table 5). Each combination, namely, BN1 and BN3, and BN2 and BN4, is present along the diagonal at the bottleneck in the main channel of the catalase. The b/a ratio of BLC was highest among the clade 3 catalases used in this study. This is most likely because residues similar in size to Val73 and Pro128 are also present in clade 1 catalases. The size of the residue Val73 is not lower than those of the corresponding residues in clade 3. Therefore, the interaction between Val73 and Phe152 in BLC, rather than between Pro60 and Phe138 in MLC, is more suitable for accepting peracetic acid as the substrate (Figs. 1, 2; Table 5).
Fig. 1.
Amino acid sequence alignment EKTA, PSCF, Deinococcus radiodurans KatA, BLC, MLC and PKTA. The amino acid resides involved in the main channel are highlighted in gray. The amino acid resides involved in the bottleneck of the main channel are surrounded by solid line
Fig. 2.
Structures of the main channels of EKTA (PDB ID:2J2M) (a), BLC (PDB ID:4BLC) (b) and MLC (PDB ID: 1GWE) (c). The accessible surfaces are represented as yellow lattices. The most important residues in the bottleneck site are underlined
Table 5.
Structural alignment of main heme channel clade 1 and clade 3 catalases used in this study
| Bottle neck no. | BN1 | BN2 | BN3 | BN4 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Clade | MC no. | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 |
| 1 | EKTA | V55 (139.1)a | H56 | V97 | D109 (117.1) | P110 (123.1) | N129 | F134 | F135 | F142 | M145 (167.7) | V146 (139.1) | L149 (164.6) | M167 (167.7) | I180 (164.9) |
| 1 | PSCF | V77 | H78 | V118 | D130 | P131 | N150 | F155 | F156 | F163 | M166 | V167 | F170 (193.5) | F188 (193.5) | L201 (164.6) |
| 1 | D. radiodurans KatA | V80 | H81 | V127 | D139 | P140 | N159 | F164 | F165 | F172 | L175 | I176 164.9) | Q179 (149.4) | F197 | L210 |
| 3 | BLC | V73 | H74 | V115 | D127 | P128 | N147 | F152 | F153 | F160 | F163 | I164 | Q167 | W185 (231.7) | L198 |
| 3 | MLC | P60 (123.1) | H61 | V101 | D113 | V114 (139.1) | N133 | F138 | F139 | F146 | F149 (193.5) | I150 | Q153 | W171 | L184 |
| 3 | PKTA | M64 (167.7) | H65 | V106 | D118 | I119 (164.9) | N138 | F142 | F143 | F151 | L154 (164.6) | N155 (127.5) | V158 (150.6) | W176 | T189 (120.0) |
aThe numbers in the transparency indicate volume (Å3) of amino acid resides in protein [29]. The important residues in EKTA and PKTA are underlined
Phylogenetic Tree of Catalases
The phylogenetic positions of the catalases used in this study were estimated based on amino acid sequences (Fig. 3). It is known that pathogenic or symbiotic microorganisms such as Neisseria gonorrhoeae and Bacteroides fragilis contain a clade 3 catalase as the sole catalase [1]. In contrast, other microorganisms possess catalases from two different clades of Bacillus subtilis and Pseudomonas putida. The above information indicated that symbiotic microorganisms live in limited niches and that the substrate for catalases is limited to H2O2. On the other hand, microorganisms that possess multiple catalases are distributed in various habitats and might interact with organisms that produce different substrates or are present in environments rich in various substrates. There was a large difference in the b/a ratio (Table 4) between the extended branches (EKTA versus PKTA) of the phylogenetic tree based on the amino acid sequence of each clade 1 and clade 3 catalase and vice versa (Fig. 3; Table 4).
Fig. 3.
Phylogenetic positions of EKTA, PSCF, Deinococcus radiodurans KatA, BLC and MLC. For construction using the maximum-likelihood method based on the JTT matrix-based model [20], multiple alignments of the sequences were performed using the MUSCLE program [19]. The numbers at the branches indicate bootstrap percentages based on 500 replicates. Bar, 0.20 changes per amino acid position. The amino acid sequences of catalases used for the alignment are shown with their NCBI database accession numbers in parentheses
Amino Acid Sequence Analysis in the Database
The possibility of an amino acid residue to be Trp185 (as in BLC) was determined. Among them, the sequence of 321 catalase-encoding genes were identified to be those of clade 3 and clade 1 catalases. Out of the 321 catalase gene sequences, 49 (15.3%) belonged to those of clade 1 catalases, while 272 (84.7%) belonged to those of clade 3 catalases. The corresponding amino acid residues in clade 1 were potentially Phe (46.9% of the 49 catalase gene sequences [size: 193.5 Å3]) followed by Met (24.5% [size: 167.7 Å3]) and Leu (6.1%). On the other hand, in clade 3, the amino acids were Trp (50.0% of the 272 clade 3 catalases [size: 231.7]), followed by Leu (24.3%), Phe (8.1%), and His (8.1%). The results described above suggested that the size of the amino acid residue at the bottleneck tended to correspond to that of Trp185 in BLC of clades 1 and 3 catalases.
Discussion
On the basis of the present study, we posit that PKTA has evolved as a highly efficient enzyme, which is consistent with its specialization in reactions with H2O2. On the other hand, EKTA has evolved as a highly efficient enzyme that accepts not only H2O2 but also larger organic peroxides. This difference may be attributed to the differences in substrate accessibility between clade 1 and clade 3 catalases. The involved clade difference might be related to the taxonomic distributions in each clade catalase. One of the reasons for the pronounced environmental adaptability is the presence of enzymes that exhibit high catalytic efficiency. E. oxidotolerans possesses a catalase (EKTA) that constitutes 6.5% of the cell extract. P. piscatorii possess a catalase (PKTA) that constitutes 10% of the cell extract. Therefore, EKTA and PKTA were not only catalytic intensities of the catalases but also high expressed levels.
Although the genera Exiguobacterium and Psychrobacter exhibit high adaptability to harsh cold environments, they differ completely in terms of phylogeny. Exiguobacterium belongs to Firmicutes, whereas Psychrobacter belongs to Proteobacteria. The wastewater drain in the food’s factory was not only cold, but also a highly oxidative environment. We believed that the ability to repress oxidative stress and tolerance to freeze–thaw stress are related [23]. The ability of both Exiguobacterium spp. and Psychrobacter to eliminate H2O2 supports their survival in not only cold freezing environments but also in the oxidative environment where high concentrations of H2O2 (6–38 mM H2O2) are existed, such as in the cold surrounding food processing factory [6, 7, 24].
Most of the amino acid residues constituting the bottleneck are highly conserved among bacterial catalase, which correlates with the high activity of catalases. However, the amino acid residues corresponding to Phe163, Ile164, Gln167 (BN2), Trp185 (BN3) and Leu198 (BN4) in BLC are not conserved as they vary with the catalases. These residues are located in the upper part of the main channel (Fig. 2), which indicates that the inward facing part of amino acid residues are critical for the appropriate catalase reaction, which includes the molecular ruler effect, interaction between charged residues and heme group, molecular motion of the amino acid side chains, and positioning of H2O2 [25]. Three among the four amino acid residues in the bottleneck of the main channel of catalases, namely, Gln167 (BN2), Trp185 (BN3), and Leu198 (BN4), vary with catalases. According to b/a ratio (Table 4), the volume of the residue corresponding to Gln167 (BN2) and Leu198 (BN4) did not have critical role in the accessibility of a large substrate. Therefore, variation in the residue corresponding to Phe163, Ile164, and Trp185 (BN3) in BLC is important for defining the accessibility of substrates larger than H2O2. Among the clade 3 catalases studied, PKTA exhibited high catalytic efficiency [15]. In addition to the narrow bottleneck structure in the main channel, the combination of Met64 and Ile119 in PKTA, which corresponded to Val73 and Pro128 in BLC, may explain this phenomenon. The combination of Met64 and Ile119 in PKTA is observed only in clade 3 catalases. The volume of this amino acid combination is larger than that in other catalases. The Met–Ile combination has been detected in catalases that exhibit high reaction efficiency with H2O2 such as V. rumoiensis [26], Proteus mirabilis and Vibrio salmonicida [27]. Therefore, the combination of Met and Ile, which correspond to Met64 and Ile119 of PKTA, which is larger in volume than the combination in BLC may contribute to reaction efficiency that is higher than those of other clade 3 catalases.
Therefore, in addition to the previously used catalase distinction between clade 1 and clade 3 catalases based on the catalytic property of substrate accessibility was investigated using PSCF, a PSCF mutant and D. radiodurans KatA [28] as clade 1 catalases and PKTA as a clade 3 catalase in the present study. The presented results indicated differences in the ratio of the compound I formation rate with peracetic acid to catalase activity with H2O2 between the extended branches in the phylogenetic tree in both clade 1 and clade 3 catalases (Fig. 3). The present study demonstrated that the clade 1 catalases exhibited considerably higher b/a ratio (0.31–1.4; Table 1) than the clade 3 catalases (0.014–0.068). Thus, the ratios indicated that the size of the amino acid residue (clade 1:164.7–193.5 Å3; clade 3:231.7 Å3) strongly correlated with substrate selection. Although the differences in the catalytic properties of clade 1 and clade 3 catalases were not clear earlier [15], for the first time, this study suggested that such differences exist, which is indicative of the differences in the molecular evolution of the two clades of catalases.
Acknowledgements
We thank Professor C Loewen (University of Manitoba, Canada) for providing catalase-less Escherichia coli UM255 harboring PSCF in pEC3E56.
Compliance with ethical standards
Conflict of interest
The authors declare that there are no competing interests.
Footnotes
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