Comparative structural and functional studies on chlorite dismutases and DyP-type peroxidases

Background.

Chlorite dismutases (Cld) are heme b containing enzymes which were discovered in chlorate- and perchlorate-reducing bacteria (van Ginkel et al., 1996) but are found in many other bacterial and archaeal phyla (Maixner et al., 2008). Reduction of (per)chlorate leads to generation of chlorite, which is a strong oxidant and has cell-damaging effects. The metalloenzyme Cld protects the organism from the accumulation of harmful chlorite by degrading it to chloride and dioxygen (van Ginkel et al., 1996). This catalytic function turns Cld into a highly interesting enzyme for bioremediation as the serious environmental pollutants chlorate and chlorite are used as bleaching agents in the textile, pulp and paper industries, as disinfectants, in pesticides etc. Additionally, Cld is extremely interesting from a biochemical point of view as it is the only known enzyme system which efficiently catalyzes O-O bond formation beside photosystem II. However, the catalytic mechanism of chlorite degradation and O2 formation by Cld is not fully understood yet (Lee et al., 2008). Moreover, the enzymatic activities and physiological roles of the vast majority of the Cld-like proteins are completely unknown. There seems to be an unexpected broad structural and functional diversity of these metalloproteins that is not understood yet (Kostan et al., 2010; Mlynek et al., 2011). Understanding these differences and the clarification of underlying reaction mechanism(s) are prerequisites for a successful application in biotechnology.

For this reason, Clds from Bradyrhizobium japonicum (Proteobacteria) and Cyanothece sp. (Cyanobacteria) were chosen and will be comprehensively investigated to gain further insight into evolution and the intrinsic biological function of these highly interesting enzymes. Moreover, Clds are phylogenetically related with a recently discovered new heme peroxidase family called Dyp-type peroxidases. Its first representative could be isolated from the fungus Thanatephorus cucumeris and was shown to have a unique tertiary structure with a distal heme region that is different from conventional peroxidases from plants and animals (Sugano et al., 2007). It was interesting to note that Clds and Dyp-type peroxidases are phylogenetically related (it is assumed that they have a common ancestor) and share a similar subunit structure as well as architecture of the heme cavity (Goblirsch et al., 2011).

Aims and methods.

This work aims to study the biochemical/biophysical properties as well as elucidate reaction mechanism(s)of the chosen enzymes. Furthermore, the intention is to get insight into the most probably divergent evolution of Clds and Dyp-type peroxidases, to investigate a possible interconversion and to attemptthe identification of a common ancestor.

• Heterologous expression and purification in E. coli.
• Site-directed and saturation mutagenesis
• Detailed spectroscopic analyses (UV/VIS spectroscopy; ECD spectroscopy; resonance Raman spectroscopy in cooperation with Giulietta SMULEVICH from the Department of Chemistry, University of Florence, Italy; electron paramagnetic resonance spectroscopy)
• Spectroelectrochemical studies (in cooperation with Gianantonio BATTISTUZZI from the Department of Chemistry, University of Modena and Reggio Emilia, Italy)
• Pre-steady-state and steady-state kinetics
• X-ray crystallography (in cooperation with Kristina DJINOVIC-CARUGO from the Department for Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna)
• Determination of conformational and thermal stability and unfolding pathway(s) (differential scanning calorimetry, temperature- and denaturant-mediated unfolding studies monitored by UV-Vis, fluorescence and ECD-spectroscopy
• Molecular modeling in cooperation with Chris OOSTENBRINK from the Institute of Molecular Modeling and Simulation at BOKU

van Ginkel, C.G., Rikken, G.B., Kroon, A.G.M., Kengen, S.W.M. (1996) Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme. Archives of Microbiology, 166, 321-326
Maixner, F., Wagner, M., Lucker, S., Pelletier, E., Schmitz-Esser, S., Hace, K., Spieck, E., Konrat, R., Le Paslier, D., Daims, H. (2008) Environmental genomics reveals a functional chlorite dismutase in the nitrite-oxidizing bacterium Candidatus Nitrospira defluvii. Environ Microbiol, 10, 3043-3056
Lee, A.Q., Streit, B.R., Zdilla, M.J., Abu-Omar, M.M., DuBois, J.L. (2008) Mechanism of and exquisite selectivity for O-O bond formation by the heme-dependent chlorite dismutase. Proc Natl Acad Sci U S A, 105, 15654-15659
Kostan, J., Sjoblom, B., Maixner, F., Mlynek, G., Furtmuller, P.G., Obinger, C., Wagner, M., Daims, H., Djinovic-Carugo, K. (2010) Structural and functional characterisation of the chlorite dismutase from the nitrite-oxidizingbacterium Candidatus Nitrospira defluvii: identification of a catalytically important amino acid residue. J Struct Biol, 172, 331-342
Mlynek, G., Sjoblom, B., Kostan, J., Fureder, S., Maixner, F., Gysel, K., Furtmuller, P.G., Obinger, C., Wagner, M., Daims, H., Djinovic-Carugo, K. (2011) Unexpected diversity of chlorite dismutases: a catalytically efficient dimeric enzyme from Nitrobacter winogradskyi. J Bacteriol, 193, 2408-2417
Sugano, Y., Muramatsu, R., Ichiyanagi, A., Sato, T., Shoda, M. (2007) DyP, a unique dye-decolorizing peroxidase, represents a novel heme peroxidase family: ASP171 replaces the distal histidine of classical peroxidases. J Biol Chem, 282, 36652-36658
Goblirsch, B., Kurker, R.C., Streit, B.R., Wilmot, C.M., DuBois, J.L. (2011) Chlorite dismutases, DyPs, and EfeB: 3 microbial heme enzyme families comprise the CDE structural superfamily. J Mol Biol, 408, 379-398