Creation of thermally tolerant peroxidase transforming a protein from thermophilic bacterium
Figure 1. Crystal structure of Cytochrome c552 (Cyt c552). PDB code: 1C52
Proteins are generally considered to be fragile under conditions other than room temperature and near neutral aqueous solution and readily lose their functions. This is because proteins familiar to us derive from cells that work in the environment close to our cells. Proteins produced in microorganisms which grow (as viewed from us) in harsh environments, however, has excellent properties to be tolerant to the environment. For example, Thermus thermophilus HB8 that we exploit in our research was found in the fumaroles of hot springs located in a rural Izu. Proteins deriving from such bacterial cells are stable at a temperature close to 100°C and can function without the degeneration. In addition, excellent durability towards organic solvents and detergents overturn the idea of "Protein is fragile and not applicable". In fact, many advanced researches in various fields with thermophilic bacteria and their proteins have been reported (Ref. 1). Proteins with various functions still lie in the thermophilic bacteria. Biological screening of properties for each protein is a method to find useful proteins. On the other hand, maintaining and excellent properties of the proteins from thermophilic bacteria, we can create a novel functional protein with the power of chemistry. The advantages of using proteins from thermophilic bacteria as a scaffold of artificial enzyme are…
- Often readily available as a recombinant proteins of E. coli, allowing a large amount of protein.
- Proteins are stable even at high temperatures. Therefore, purification and handling is easy.
- Against multiple mutagenesis, the proteins retains the original conformation. Therefore, functional design is easy.
1) and 2) are likely to be unrelated with essence of studies. However, easy purification and handing of the product is very important points for day-to-day experiments. In the following, we briefly introduce our research using proteins from thermophilic bacteria.
Figure 2. Active site of the Cyt c552 mutant designed by mutagenesis.
Among the proteins derived from Thermus thermophilus, we chose cytochrome c552 (Cyt c552, Figure 1) which is an small electron transfer having heme as aprosthetic group. Three-dimensional structure of this protein has been already resolved. Using this protein as a scaffold, we attempted to produce an artificial thermally tolerant peroxidase. The general acid-base catalyst is the key feature of peroxidase reaction (Figure 2). The electron transfer protein, Cyt c552 has no such mechanism originally. Therefore, we introduced it in the vicinity of the heme by mutagenesis to incorporate a catalytic
Figure 3. Peroxidase activity of the V49D/M69A mutant compared with that of the Mb H64D. ¡: V49D/M69A, △: Mb H64D. (a) The temperature dependence of the catalytic activity. Initial turnovers at 0 min are compared. (b) Time course of substrate consumption at 70ºC. Reaction conditions: substrate (ferulic acid) 200 m M; protein 0.2 m M; H2O2 200 mM, 20 mM MES-MaOH buffer solution (pH 5.0).
mechanism in the heme cavity. Based on the three-dimensional structure of the protein, I was replaced Val49 with Asp which is expected to place at approximately 5.2 Å above the heme iron. Heme of Cyt c552 has six-octahedral coordination structure and there is no coordination sites for reactions. Therefore, Met69 which is one of the original ligand was replaced with Ala to form the vacant site on the iron ion. Figure 2 shows schematic view of the vicinity of heme (V49D/M69A) mutant. Figure 3 shows the peroxidase activity of this mutant (Figure 3a,¡). The catalytic activity of the variant increases as temperature rises. When compared with the H64D mutant of myoglobin which has been reported previously to has highest peroxidase activity so far (Figure 3a, △), Mb H64D shows higher activity than our mutant up to 40 º C. However, at 50ºC both are comparable and at 60 and 70 º C, the V49D/M69A mutant suppresses the myoglobin mutant. Rapid decrease in activity at 80ºC is associated with formation of hexacoordinated heme species produced by an unidentified ligand, which blocks the active site although the mutant keeps intact without denaturation. Therefore, the catalytic activity comes out again at slightly lower temperature the 80ºC. Superiority of the V49D/M69A mutant under high temperature conditions is also seen on the sustainability of the activity. Figure 3b shows the time course of substrate consumption observed when using the Mb H64D and V49D/M69A mutants. The peroxidase reaction was carried out at 70ºC. The V49D/M69A mutant always exceeds the myoglobin mutant in the amount of substrate consumption. This demonstrates that even in the sustainability of the catalytic activity, our mutant is superior to the myoglobin mutant. These results have shown that the original thermal stability and durability of the Cyt c552 are varied for a high catalytic activity and persistence in the high temperature region.
- Thermophiles Biodiversity, Ecology, and Evolution, A. –L. Reysenbach, M. Voytek, and R. Mancinelli Ed., Kluwer Academic/Plenum Publishers, 2001, NY.
Our paper concerned with this study）
- Cytochrome c552 from Thermus Thermophilus Engineered for Facile Conversion of the Prosthetic Group, Biochemistry Accepted, Ibrahim, Sk. Md., Nakajima, H., Ramanathan, K., Takatani, N., Ohta, T., Naruta, Y., Watanabe, Y.
Because of a covalent bonding between the protein backbone and a prosthetic group, Replacement of the heme from the protein is bothersome and time consuming process, and the yield of the reconstituted cytochrome is generally low. We have developed a novel system that provides a facile method to replace the original heme to an artificial molecule and subsequent covalent bond formation between the protein backbone and the artificial prosthetic group by exploiting Cyt c552 from thermophilic bacterium,
- Molecular Design of Heme Proteins for Future Application, Catal. Surv. Asia 2011, 15, 134-143. Nakajima, H., Shoji, O., Watanabe, Y.
- Rational engineering of Thermus thermophilus Cytochrome c552 to thermally tolerant artificial peroxidase, J. Chem. Soc. Dalton Trans. 2010, 39, 3105-3114, Nakajima, H., Ramanathan, R., Kawaba, N., Watanabe, Y.
Cyt c552 from thermophilic bacterium can be transformed to peroxidase which functions even at high temperatures (<80ºC). A fatal problem of the thermally tolerant peroxidase is rapid inactivation during the catalytic reaction. In this study, we attempted to develop the stability of the artificial peroxidase in the catalytic cycle by evaluating the detailed inactivation mechanism. The results supports validity of rational design of artificial enzymes based on chemistry behind the catalyst.
- Engineering of Thermus thermophilus Cytochrome c552: Thermally Tolerant Artificial Peroxidase, ChemBioChem 2008, 9, 2954-2957, Nakajima H., Ichikawa Y., Satake Y., Takatani N., Manna SK., Rajbongshi J., Mazumdar S., Watanabe Y.
- Reactivities of oxo and peroxo intermediates studied by hemoprotein mutants., Acc Chem Res 2007, 40, 554-562, Watanabe Y., Nakajima H. and Ueno T.
- Characterization of peroxide bound heme species generated in reaction of thermally tolerant cytochrome c552 with hydrogen peroxide. ChemBioChem 2006, 7, 1582-1589, Ichikawa Y., Nakajima H., Watanabe Y.
A highly hydrophobic cavity furnished in a Cyt c552 variant affords a transient intermediate, hydroperoxo-ferric heme species in reaction with hydrogen peroxide under ambient conditions. The obtained hydroperoxo-ferric heme species is in pre-equilibrium state and has sufficient stability to characterize its reactivity in a peroxidase reaction cycle.