Most bacteria, yeasts, fungi, algae, and higher plants use inorganic sulfate as their sole sulfur source for the biosynthesis of cysteine, methionine, all reduced organic sulfur-containing coenzymes (biotin, thiamin, coenzyme A, lipoate, etc.), and a variety of structural components (plant sulfolipid, sulfated polysaccharides, etc.). The overall objective of the research is to establish the catalytic mechanism, regulatory properties, and structure-function relationships of the enzymes which catalyze the earliest steps of inorganic sulfate assimilation by fungi. Thus, a study of the enzymes (a) ATP sulfurylase, (b) APS kinase, and (c) "PAPS reductase" which catalzye, in order, (a) the formation of adenosine-5'- phosphosulfate (APS) from SO42- and ATP, (b) the formation of 3'-phosphoadenosine- 5'- phosphosulfate (PAPS) from APS and ATP, and (c) the NADPH and thioredoxin-dependent reduction of PAPS to free or bound sulfite. "PAPS reductase" is actually a multicomponent system which has never been purified to homogeneity of kinetically characterized. Most of the experiments will be performed with enzymes purified from the mesophilic fungus, Penicillium chrysogenum. Parallel studies will be performed on the enzymes from the thermophile, Penicillium duponti with the objective of identifying the structural features responsible for the remarkable heat stability of proteins from this organism. Enzyme kinetics, equilibrium binding, protein chemistry, and molecular biology methods will be used.
