Description
The complete draft genome of the fungus Phanerochaete chrysosporium was published in June 2004 (Nat. Biotechnol. 22, 695). The fact that the first basidiomycete whose whole genome is available (http://www.jgi.doe.gov/) is a model species for lignin biodegradation studies is indicative of the interest of these organisms (and their enzymes) in different biotechnological applications. The present situation is very promising for these studies because it will permit to identify and characterize the whole set of enzymes and other proteins involved. Lignin biodegradation is a complex extracellular process involving hydrogen peroxide-producing oxidases and high redox potential peroxidases catalyzing the oxidative degradation of this polymer (“enzymatic combustion”) by the hydrogen peroxide. In the present project we will study at a molecular level the reactions catalyzed by oxidases and peroxidases recently described by our group, together with those new oxidoreductases whose genes will be identified during the analysis of the genome of P. chrysosporium. For the functional and structural analyses of the products of these new genes it will be necessary to optimize a suitable system of heterologous expression. With this purpose we will investigate both the eukaryotic expression of the whole genes with their introns, and the prokaryotic expression of cDNA (followed by in vitro activation when required). The enzymes and other proteins of interest (identified in the genome analysis or recently described by our group) will be subjected to a functional and structural characterization including, among others, studies of X-ray diffraction (general structure), nuclear magnetic resonance (NMR) when required in metalloprotein studies, rapid kinetics (catalytic cycle intermediates), steady-state kinetics (comparison of enzyme activities and affinities), macromolecular interactions (with lignin) by advanced light-scattering techniques, electronic paramagnetic resonance (EPR, detection of radicals), cyclic voltammetry (redox potential), molecular dynamics and docking (to predict the active sites and other properties), and site-directed mutagenesis (to confirm the potentially relevant amino acid residues). Finally, we will evaluate the biotechnological potential of the different oxidoreductases investigated, and the enzyme variants obtained using mutagenesis techniques, based on their activities on lignin model compounds (and other high redox potential compounds) and their stability under working conditions (pH and temperature) similar to those required for their future utilization as industrial biocatalysts.