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Dr. BarondeauResearch in the Barondeau lab is focused on determining structure-function properties of proteins and enzymes and elucidating chemical mechanisms that underlie biological function. We utilize X-ray crystallography as a primary tool for discovery-based science followed by the development and testing of specific hypotheses using molecular biology, biochemistry, spectroscopy, and other biophysical techniques. In particular, we seek a detailed understanding of proteins/enzymes involved in eukaryotic Fe-S cluster biogenesis, UV resistance in sporylating pathogens, and chlorophyll biosynthesis. Eukaryotic Fe-S cluster biogenesis. In eukaryotes,
Fe-S cluster biosynthesis is initiated in the mitochondrial matrix through a
complex metallochaperone system that includes at least 10 proteins and a
choreographed network of protein-protein interactions and complexes. Fe-S
clusters are generated on scaffolding proteins via protein-based transfer of
sulfur, iron, and electrons, followed by chaperone-mediated delivery of intact
clusters to their target proteins. To investigate this Fe-S assembly pathway, we
employ a novel thermophilic eukaryotic model system, the aerobic sea worm
Alvinella pompejana that thrives on black smoker chimneys 2500 meters under the
ocean surface. At room or low temperature, thermophilic homologs tend to be more
rigid, favoring crystallization and X-ray structural analyses, and have much
slower UV resistance in sporylating pathogens. Pathogenic bacteria are often difficult to destroy due to their ability to form highly stable and persistent spores that are resistant to heat, acid, hydrogen peroxide, and UV-induced DNA damage. The UV resistance of endospore-forming bacteria is due to two primary factors. First, UV exposure in dormant spores results in the formation of thymine dimer 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)] DNA damage (Fig. 1), rather than the cyclobutane-like dimer observed during vegetative growth. This unusual DNA photochemistry results from the binding of small, acid-soluble spore proteins that alter the helical conformation of DNA. Second, this DNA damage is repaired early in spore germination independent of de novo protein synthesis, suggesting the SP repair enzymes are packaged in the dormant spore. The homodimeric SP lyase repairs these DNA lesions by utilizing a [Fe4S4] cluster to split S-adenosyl methionine and initiate radical-based chemistry. SP lyase is found in sporylating pathogens such as Bacillus anthracis, Clostridium botulina, and Clostridium perfringes (“flesh-eating bacteria) and gene deletion results in spores more susceptible to UV-induced DNA damage and cell death. Here our objectives are to understand the structure and mechanism of SP lyase and the remarkable UV-resistance for these sporylating pathogens.
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enzymatic turnover, permitting temperature trapping of intermediates and
protein-protein interactions. Here our objectives are to define intermediate
states and the mechanism for Fe-S clusters synthesis, target recognition, and
transfer. Defects in Fe-S cluster biosynthesis are directly associated with
human diseases such as Friedreich's ataxia and X-linked sideroblastic ataxia
(ataxia is the inability to coordinate muscular movement). 
Chlorophyll
biosynthesis. Photosynthetic organisms are responsible for O2 production and
CO2 assimilation that define the composition of our atmosphere, and for
producing the food and fuel necessary for most life on earth. Chlorophyll
pigments are essential for photosynthesis, yet surprisingly little is known
about their biosynthesis. Here we seek to determine structure-function
properties for two intriguing enzymes that catalyze the protochlorophyllide
reduction (por) step in chlorophyll biosynthesis. The dark-adaptive dpor may
have evolved from nitrogenase and reduces its substrate in a ATP-driven process
using an uncharacterized Fe-S complex (Fig. 2). Light-dependent lpor is a
NAD(P)H oxidoreductase that uses an unusual photochemical reaction to achieve
the same chemistry. Here our objectives are to elucidate structural details for
the por enzymes from the thermophilic cyanobacteria T. elongatus and address
detailed mechanistic questions for this critical step in chlorophyll
biosynthesis.