Structure-Function Studies on the Sulfate Activating Enzymes

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This research project focuses on the structural features underlying the catalytic and regulatory properties of the first sulfate activating enzyme, ATP sulfurylase. ATP sulfurylase plays multiple roles in nature: (a) In fungi, yeasts, most heterotrophic bacteria, algae, and higher plants, this enzyme catalyzes the first intracellular reaction in the reductive assimilation of inorganic sulfate into organic molecules. In these organisms, APS is the active sulfate precursor of cysteine, methionine, etc. In all organisms, PAPS serves as the sulfuryl donor for sulfate ester biosynthesis. (b) In anaerobic sulfate reducing bacteria (e.g., Desulfovibrio), ATP sulfurylase forms APS solely to serve as the terminal electron acceptor of heterotrophic metabolism. (c) In certain chemo- and photolithotrophic bacteria (e.g., Aquifex, Chromatium), ATP sulfurylase catalyzes the last reaction in the oxidation of reduced inorganic sulfur compounds to sulfate i.e., the physiological reaction is in the opposite direction compared to that in sulfate assimilators. Sulfate (sulfuric acid) produced by sulfur chemolithotrophs is believed to be responsible for shaping many terrestrial caverns and for the acidic runoff from pyrite-containing mines. A long-term goal of this project is to identify the structural features that optimize the catalytic and regulatory properties of each class of homooligomeric ATP sulfurylase for the physiologically relevant task. Immediate efforts will focus on the homohexameric enzyme of Penicillium chrysogenum, which is allosterically inhibited by PAPS, the product of the APS kinase-catalyzed reaction. Experiments are proposed to determine (a) the mechanism by which the APS kinase-like regulatory domain communicates with the catalytic domain, (b) the structural basis for the high selectivity of the catalytic and allosteric sites for sulfate/sulfonucleotide over the more common phosphorous analogs, and (c) the functional properties of several chimeric forms including one that contains true APS kinase as the C-terminal domain and may channel APS between sites.Broader Impacts: A cross section of California students will receive training in the methods of molecular biology (cloning, site-directed mutagenesis, expression and purification of mutant and chimeric enzymes), enzymology (steady-state kinetics, equilibrium ligand binding, computer-assisted simulation and analysis of data), and protein chemistry (x-ray crystallography, chemical modification of target residues). The experiences help to prepare students for advanced academic programs or for entry-level positions in biotechnology firms. Students from underrepresented groups participate in our research as part of the Biology Undergraduate Scholars Program (BUSP) and Minority Research Participation in the Physical Sciences Program (MRPPSP).