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Browsing by Author "Fedkenheuer, Michael Gerald"

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    Structural and Mutational Analyses of Aspergillus fumigatus SidA: A Flavin-Dependent N-hydroxylating Enzyme
    Fedkenheuer, Michael Gerald (Virginia Tech, 2012-07-27)
    SidA from Aspergillus fumigatus is an N-hydroxylating monooxygenase that catalyzes the committed step in siderophore biosynthesis. This gene is essential for virulence making it an excellent drug target. In order to design an inhibitor against SidA a greater understanding of the mechanism and structure is needed. We have determined the crystal structure of SidA in complex with NADP+, Ornithine, and FAD at 1.9 ? resolution. The crystal structure has provided insight into substrate and coenzyme selectivity as well as residues essential for catalysis. In particular, we have chosen to study the interactions of Arg 279, shown to interact with the 2'phosphate of the adenine moiety of NADP+ as well as the adenine ring itself. The mutation of this residue to alanine makes the enzyme have little to no selectivity between coenzymes NADPH and NADH which supports the importance of the ionic interaction between Arg279 and the 2'phosphate. Additionally, the mutant enzyme is significantly more uncoupled than WT enzyme with NADPH. We see that the interactions of the guanadinyl group of Arg279 and the adenine ring are also important because KM and Kd values for the mutant enzyme are shifted well above those of wild type with coenzyme NADH. The data is further supported by studies on the reductive and oxidative half reactions. We have also explored the allosteric effect of L-arginine. We provide evidence that an enzyme/coenzyme/L-arginine complex is formed which improves coupling, oxygen reactivity, and reduction in SidA; however more work is needed to fully understand the role of L-arginine as an allosteric effector.
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    Understanding Plant Pathosystems in Wild Relatives of Cultivated Crop Plants
    Fedkenheuer, Michael Gerald (Virginia Tech, 2016-08-09)
    As the global population rises, the demand for food increases which underscores a need for improvement in food security. Disease pressures are a major concern surrounding sustainable agriculture. Static crop populations, containing little to no genetic diversity, are vulnerable to diverse pathogen populations. Wild relatives of crop plants are a reservoir for new disease resistance traits that can be introgressed into cultivated crops. The identification of novel disease resistance is of paramount importance because pathogen co-evolution is not only defeating current resistance genes (R genes) but chemical controls as well. Phytophthora sojae (P. sojae), the causal agent of Phytophthora root and stem rot disease, reduces soybean harvests worldwide. We developed an approach to screen for new R genes that recognize core effectors from P. sojae. We expect R genes identified by these screens to be durable because P. sojae requires core effectors for virulence. We utilized effector-based screening to probe Glycine soja germplasm with core RXLR effectors from P. sojae to search for novel R genes. We developed segregating populations from crosses of P. sojae resistant G. soja germplasm with susceptible G. max cultivar Williams to determine inheritance of potential R genes in germplasm that responded to core effectors. We are using marker assisted breeding to map disease resistance traits in recombinant inbred (RI) lines. To better understand pathosystems, we examined host resistance and susceptibility using bioinformatics. We analyzed the interaction between Arabidopsis thaliana ecotype Col-0 and Hyaloperonospora arabidopsidis isolate Emwa1 using a publicly available RNA time-course experiment. We describe a new algorithm to sort genes into time-point specific clusters using activation and repression parameters. Gene ontology annotations were used to identify defense genes with unique expression profiles, and A. thaliana null mutants for these genes were significantly more susceptible to Emwa1 than wild-type. We plan to use these tools to rapidly identify and guide introgression of durable disease resistance into crop species.