main infectious agents in this regard are the opportunistic pathogens Candida albicans (responsible for candidiasis) and AspergiUus fumigatus (responsible for aspergillosis), and Cryptococcus neoformans (responsible for cryptococcosis). The aminoadipate pathway for the biosynthesis of lysine in fungi is for the most part unique to these organisms. The seven-enzyme pathway has been studied to a limited extent, but all of the reactions have mechanistic precedence in other systems. The PI initially proposes to study the regulated homocitrate synthase and the saccharopine dehydrogenases, which catalyze the final two steps in the pathway. Each of the enzymes will be studied to determine their mechanism with respect to transition state structure. Studies will be conducted according to the following specific aims. 1) The gene for the lysine-regulated homocitrate synthase has been obtained, expressed, purified and characterized. The genes for the glutamate- and lysine-forming saccharopine dehydrogenases have been obtained by PCR from the Saccharomyces cerevisiae genome, and are being subcloned into the pQE expression vector (Qiagen). They will then be expressed in Escherichia coli, and purified. 2) Each of the three enzymes will be crystallized in the absence and presence of appropriate ligands. One of the enzymes, saccharopine dehydrogenase (Lglutamate forming) from Magnaporthe grisea has been crystallized and had its structure solved in the presence of substrates. 3) Each of the three enzymes will be studied with respect to their kinetic, and catalytic mechanism using a variety of techniques including steady state and presteady state kinetic studies, measurement of pH-rate profiles and isotope effects, and site-directed mutagenesis. The only enzyme that has been studied mechanistically to any extent is the final enzyme in the pathway, saccharopine dehydrogenase (L-lysine forming), and these data will provide a starting point for further, more complete studies. 4) Site-directed mutagenesis will be carried out initially on saccharopine dehydrogenase (Lglutamate forming). Three residues have been selected as possible catalytic residues, and these will be changed and the resulting mutants will be characterized. Additionally, possible catalytic and binding residues in the homocitrate synthase have been identified by multiple sequence alignment, and alanine scanning mutagenesis will be applied to these.

R01GM071417

2004-09-15

2009-08-31