Abstract

Bacteria and fungi are able to grow using diverse aromatic compounds as sole sources of energy and carbon (1) (2) . These abilities are of interest since many of these compounds are toxic to higher organisms, which cannot degrade them. While bacterial enzymes responsible for degradation of aromatics have been quite well studied, relatively little is known about their fungal counterparts. The aim of this thesis is to expand knowledge of aromatic compound degradation in fungi. cDNA libraries from 15 diverse fungal species are available from the Concordia fungal genomics group and within this database there are many BLAST (Basic Local Alignment Search Tool) hits to genes encoding enzymes involved in aromatic degradation pathways. Using bioinformatics techniques, DNA sequences encoding putative mono- and dioxygenases that initiate aromatic compound degradation were identified. Genes encoding these enzymes were amplified and used to construct recombinant plasmids for overexpression of the corresponding proteins, which were then purified, characterized and compared to their bacterial counterparts. A putative salicylate hydroxylase from L. edodes , the shiitake mushroom, and a putative phenol hydroxylase and a catechol-1 ,2-dioxygenase from Gloeophyllum trabeum were expressed in E. coli . The putative salicylate hydroxylase was purified in good yield using two chromatographic steps. The purified protein contained a 1:1 ratio of non-covalently bound FAD (flavin adenine dinucleotide). Interestingly, salicylate was a non-substrate effector, stimulating production of hydrogen peroxide from NADPH and oxygen, rather than hydroxylated product formation. In contrast, 2-aminobenzoate was rapidly converted to 2,3-dihydroxbenzoate. Furthermore, spectroscopic probes of binding indicated that the modes of binding of salicylate and 2-aminobenzoate are quite different. Together, these results suggest that the enzyme is not a salicylate hydroxylase, and that the true substrate is an amino aromatic compound. The putative phenol hydroxylase from G. trabeum was also purified in good yield using two chromatographic steps and was found to contain 1 mol of FAD per mol of protein. Uncoupling assays and product analysis results suggest that the enzyme is a resorcinol-specific hydroxylase, while phenol is a non-substrate effector. The putative catechol-l,2-dioxygenase was purified using four chromatographic steps, however, no activity was observed under any condition. In summary, potential functions were elucidated for two new fungal flavoprotein hydroxylases, whose activities were not accurately predicted using sequence comparisons. Additional work will be required to further examine the activities of the putative catechol 1,2-dioxygenase.