Unique presence of a manganese catalase in a hyperthermophilic archaeon, Pyrobaculum calidifontis VA1

Abstract

We had previously isolated a facultatively anaerobic hyperthermophilic archaeon, Pyrobaculum calidifontis strain VA1. Here, we found that strain VA1, when grown under aerobic conditions, harbors high catalase activity. The catalase was purified 91-fold from crude extracts and displayed a specific activity of 23,500 U/mg at 70 degrees C. The enzyme exhibited a K(m) value of 170 mM toward H(2)O(2) and a k(cat) value of 2.9 x 10(4) s(-1).subunit(-1) at 25 degrees C. Gel filtration chromatography indicated that the enzyme was a homotetramer with a subunit molecular mass of 33,450 Da. The purified catalase did not display the Soret band, which is an absorption band particular to heme enzymes. In contrast to typical heme catalases, the catalase was not strongly inhibited by sodium azide. Furthermore, with plasma emission spectroscopy, we found that the catalase did not contain iron but instead contained manganese. Our biochemical results indicated that the purified catalase was not a heme catalase but a manganese (nonheme) catalase, the first example in archaea. Intracellular catalase activity decreased when cells were grown anaerobically, while under aerobic conditions, an increase in activity was observed with the removal of thiosulfate from the medium, or addition of manganese. Based on the N-terminal amino acid sequence of the purified protein, we cloned and sequenced the catalase gene (kat(Pc)). The deduced amino acid sequence showed similarity with that of the manganese catalase from a thermophilic bacterium, Thermus sp. YS 8-13. Interestingly, in the complete archaeal genome sequences, no open reading frame has been assigned as a manganese catalase gene. Moreover, a homology search with the sequence of kat(Pc) revealed that no orthologue genes were present on the archaeal genomes, including those from the "aerobic" (hyper)thermophilic archaea Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii. Therefore, Kat(Pc) can be considered a rare example of a manganese catalase from archaea.

FIG. 1.

SDS-PAGE of samples during the purification process of the P. calidifontis VA1 catalase. Lane M, molecular mass markers; lane 1, crude extract (5 μg); lane 2, ammonium sulfate fraction (5 μg); lane 3, peak fraction after hydrophobic-interaction chromatography (5 μg); lane 4, flowthrough fraction after anion-exchange chromatography (5 μg); lane 5, purified enzyme obtained from gel filtration chromatography (5 μg). The arrows indicate the major and minor bands described in the text.

FIG. 2.

(A) Optimum pH of the P. calidifontis VA1 catalase. The buffers used for each pH range were 50 mM acetic acid-sodium acetate buffer (pH 4.0 to 5.5, closed squares); 50 mM potassium phosphate buffer (pH 5.5 to 8.0, closed circles); 50 mM Tris-HCl buffer (pH 7.5 to 9.0, open circles); 50 mM glycine-NaOH (pH 9.0 to 9.5, closed triangles); and 25 mM sodium hydrogen carbonate-sodium carbonate buffer (pH 9.5 to 10.0, open triangles). The assay temperature was 70°C. The results are shown as percentage ratios relative to the specific activity of the catalase in 50 mM glycine-NaOH buffer (pH 9.5). (B) Optimum temperature of the P. calidifontis VA1 catalase. (C) Thermostability of the P. calidifontis VA1 catalase at various temperatures. Closed circles, incubation at 100°C; open circles, incubation at 95°C; closed triangles, incubation at 90°C.

FIG. 2.

(A) Optimum pH of the P. calidifontis VA1 catalase. The buffers used for each pH range were 50 mM acetic acid-sodium acetate buffer (pH 4.0 to 5.5, closed squares); 50 mM potassium phosphate buffer (pH 5.5 to 8.0, closed circles); 50 mM Tris-HCl buffer (pH 7.5 to 9.0, open circles); 50 mM glycine-NaOH (pH 9.0 to 9.5, closed triangles); and 25 mM sodium hydrogen carbonate-sodium carbonate buffer (pH 9.5 to 10.0, open triangles). The assay temperature was 70°C. The results are shown as percentage ratios relative to the specific activity of the catalase in 50 mM glycine-NaOH buffer (pH 9.5). (B) Optimum temperature of the P. calidifontis VA1 catalase. (C) Thermostability of the P. calidifontis VA1 catalase at various temperatures. Closed circles, incubation at 100°C; open circles, incubation at 95°C; closed triangles, incubation at 90°C.

FIG. 3.

Lineweaver-Burk plot of the P. calidifontis VA1 catalase with the substrate hydrogen peroxide. Activity measurements were performed at 25°C for comparison with other manganese and heme catalases.

FIG. 4.

Amino acid sequence alignment of Kat_Pc (Pc) with manganese catalases from Thermus sp. YS 8-13 (Th, accession no. AB008786) and L. plantarum (Lp, accession no. D87070). Alignment was performed with CLUSTAL W. Conserved residues are indicated with asterisks. Domain boundaries were determined by using the structure of the T. thermophilus catalase (3). Residues highlighted with a solid background are residues coordinated with manganese ions. The lysine (Pc and Th) or glutamate (Lp) residues indicated by arrowheads are considered to contribute to catalytic proton transfer. The calcium binding site of the catalase from L. plantarum is boxed.