Study of substrate binding sites and other aspects of the molecular architecture of novel peroxidases and oxidases involved in degradation of lignin and aromatic compounds (Funded by BIO2002-01166, 2002-05; and Agrenvec)

Extracellular oxidation by H₂O₂ (generated by oxidases) in a reaction catalyzed by high redox potential peroxidases is a key step in lignin biodegradation. The process has interest for oxidation of a variety of aromatic compounds. The applicants have cloned, characterized, and expressed in heterologous systems two enzymes that act synergistically in lignin attack by Pleurotus eryngii. These enzymes are: i) a novel per oxidase type, we called versatile peroxidase (VP) due to its ability to oxidize typical substrates of the two other ligninolytic peroxidases (i.e. lignin peroxidase and manganese peroxidase, sharing 54-61% sequence with VP); and ii) an oxidase, aryl-alcohol oxidase (AAO) producing H₂O₂ from oxidation of aromatic alcohols (sharing 34% sequence with Aspergillus niger glucose oxidase). The objective of the project is to establish their molecular architecture, with special emphasis on the substrate binding sites using different techniques (such as site-directed mutagenesis, x-ray diffraction, ¹H-NMR, MALDI-TOF, molecular modeling, circular dichroism, optical biosensor, and stopped flow). Recombinant VP (VP*) will be expressed in Escherichia coli followed by optimized “in vitro” folding, while AAO* will be initially produced in Emericella nidulans. Crystallization conditions have been obtained for both enzymes, therefore, crystal models could be soon available. VP studies will also include. i) confirmation of the role of three acidic residues forming a Mn-binding site near the internal propionate of heme (postulated from the first models and mutagenesis results); ii) identification of the sites involved in oxidation of aromatic substrates, including electron transfer pathways (one of them from an exposed Trp) or direct oxidation at the heme channel; and iii) modulate the activity on different substrates by modifying the corresponding binding sites. In the case of AAO, we plan: i) to identify the residues involved in catalysis (including two histidines near the flavin ring) and the corresponding mechanism; and ii) to establish the architecture of the substrate pocket taking into account that it is able to bind efficiently alcohols with very different molecular structures.