? 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. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM071417-03
Application #
7119238
Study Section
Biochemistry Study Section (BIO)
Program Officer
Jones, Warren
Project Start
2004-09-15
Project End
2008-08-31
Budget Start
2006-09-01
Budget End
2007-08-31
Support Year
3
Fiscal Year
2006
Total Cost
$244,867
Indirect Cost
Name
University of Oklahoma Norman
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
848348348
City
Norman
State
OK
Country
United States
Zip Code
73019
Vashishtha, Ashwani K; West, Ann H; Cook, Paul F (2015) Probing the chemical mechanism of saccharopine reductase from Saccharomyces cerevisiae using site-directed mutagenesis. Arch Biochem Biophys 584:98-106
Hsu, Chaonan; West, Ann H; Cook, Paul F (2015) Evidence for an induced conformational change in the catalytic mechanism of homoisocitrate dehydrogenase for Saccharomyces cerevisiae: Characterization of the D271N mutant enzyme. Arch Biochem Biophys 584:20-7
Kumar, Vidya Prasanna; Thomas, Leonard M; Bobyk, Kostyantyn D et al. (2012) Evidence in support of lysine 77 and histidine 96 as acid-base catalytic residues in saccharopine dehydrogenase from Saccharomyces cerevisiae. Biochemistry 51:857-66
Ekanayake, Devi K; West, Ann H; Cook, Paul F (2011) Contribution of K99 and D319 to substrate binding and catalysis in the saccharopine dehydrogenase reaction. Arch Biochem Biophys 514:8-15
Bobyk, Kostyantyn D; Kim, Sang Gon; Kumar, Vidya Prasanna et al. (2011) The oxidation state of active site thiols determines activity of saccharopine dehydrogenase at low pH. Arch Biochem Biophys 513:71-80
Ekanayake, Devi K; Andi, Babak; Bobyk, Kostyantyn D et al. (2010) Glutamates 78 and 122 in the active site of saccharopine dehydrogenase contribute to reactant binding and modulate the basicity of the acid-base catalysts. J Biol Chem 285:20756-68
Vashishtha, Ashwani Kumar; West, Ann H; Cook, Paul F (2009) Chemical mechanism of saccharopine reductase from Saccharomyces cerevisiae. Biochemistry 48:5899-907
Lin, Ying; West, Ann H; Cook, Paul F (2009) Site-directed mutagenesis as a probe of the acid-base catalytic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae. Biochemistry 48:7305-12
Lin, Ying; West, Ann H; Cook, Paul F (2008) Potassium is an activator of homoisocitrate dehydrogenase from Saccharomyces cerevisiae. Biochemistry 47:10809-15
Qian, Jinghua; Khandogin, Jana; West, Ann H et al. (2008) Evidence for a catalytic dyad in the active site of homocitrate synthase from Saccharomyces cerevisiae. Biochemistry 47:6851-8

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