Theses and Dissertations at Montana State University (MSU)
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Item Discovery of key intermediates for radical initiation in PFL-AE(Montana State University - Bozeman, College of Letters & Science, 2020) McDaniel, Elizabeth Claire; Chairperson, Graduate Committee: Joan B. Broderick; This is a manuscript style paper that includes co-authored chapters.Members of the radical S-adenosyl-L-methionine (SAM) enzyme superfamily utilize a [4Fe-4S] cluster and the small molecule, SAM, to generate methionine and the 5'deoxyadenosyl radical (5'-dAdo*). Once formed, the 5'-dAdo* abstracts a hydrogen from substrate allowing for the catalyzation of a wide array of chemistry such as DNA repair, hydrogenase maturation, and anaerobic glucose metabolism. Originally, the 5'-dAdo* was thought to form directly through homolytic cleavage of the S-C5' bond on SAM. In 2016, this mechanism was called into question when a catalytically relevant organometallic intermediate (omega) was discovered in pyruvate formate-lyase activating enzyme (PFL-AE). This intermediate consisted of a 5'-dAdo moiety bound to the unique iron on the PFL-AE [4Fe-4S] cluster through an Fe-C5' bond. The work shown in this thesis provides novel insights into the RS enzyme mechanism considering the newly discovered omega species. Using rapid freeze quench (RFQ) in conjunction with electron paramagnetic resonance (EPR) spectroscopy, omega formation was observed in seven RS enzymes representing the totality of superfamily reaction types. Inspired by the idea that the Fe-C5' bond in omega could undergo photoinitiated homolysis, a unique procedure was developed to generate and capture the long elusive 5'-dAdo* through cryogenic photolysis of reduced PFL-AE and SAM. Isotopic labeling of SAM along with EPR spectroscopy confirmed definitely that this was the long sought after 5'-dAdo*. To better understand RS enzyme bond specificity and the order of intermediate formation, an analogue of SAM, S-3'4'-anhydroadenosyl-L-methionine (anSAM), was employed in RFQ and cryogenic photolysis experiments. By using anSAM, it was shown that the bond cleavage specificity of PFL-AE can changed under appropriate conditions and provided evidence that omega forms first in the radical initiation pathway of RS enzymes. These results have greatly increased our understanding of the RS enzyme mechanism and will help future work designed to utilize the incredible enzymatic potential of this diverse superfamily.Item A molecular, structural, and cellular multiple-level study aimed at understanding the unique reaction catalyzed by the last enzyme in the heme-biosynthesis pathway of gram-positive bacteria, coproheme decarboxylase (CHDC)(Montana State University - Bozeman, College of Letters & Science, 2018) Celis Luna, Arianna I.; Chairperson, Graduate Committee: Jennifer DuBois; Bennett R. Streit, Garrett C. Moraski, Timothy D. Lash, Gudrun S. Lukat-Rodgers, Kenton R. Rodgers and Jennifer L. DuBois were co-authors of the article, 'Unusual peroxide-dependent, heme-transforming reaction catalyzed by hemQ' in the journal 'Biochemistry' which is contained within this thesis.; George H. Gauss, Bennett R. Streit, Krista Shisler, Garrett C. Moraski, Kenton R. Rodgers, Gudrun S. Lukat-Rodgers, John W. Peters and Jennifer L. DuBois were co-authors of the article, 'A structure-based mechanism for oxidative decarboxylation reactions mediated by amino acids and heme propionates in coproheme decarboxylase (hemQ)' in the journal 'Journal of the American Chemical Society' which is contained within this thesis.; Dissertation contains two articles of which Arianna I. Celis Luna is not the main author.Heme b is one of nature's most ancient and versatile co-factors and is essential for aerobic life. As such, heme b is synthesized by almost every living organism and plays a major role in bacterial virulence. A pathway for heme b biosynthesis, which is unique to some of the most primitive gram-positive bacteria including many important pathogens, was recently discovered. This pathway, now known as the coproprophyrin-dependent (CPD) branch, ends in a step catalyzed by an unusual enzyme known alternately as coproheme decarboxylase (ChdC) or HemQ. This research aimed to understand ChdC function at the molecular, structural, and cellular levels. Using the ChdC enzyme from Staphylococcus aureus (SaChdC) and a variety of biochemical and analytical tools (conventional and stopped-flow UV-Vis spectroscopy, resonance Raman, HPLC, LC-MS, site-directed mutagenesis, EPR, and X-ray crystallography), the work presented here describes how the coproheme substrate is accommodated in the SaChdC active site and poised for reactivity. The cumulative results show that ChdC catalyzes the oxidative decarboxylation of coproheme III to generate heme b in a sequential and clock-wise fashion, generating harderoheme III in the process. This reaction is H 2 O 2-dependent and the mechanism involves the formation of the high-valent Fe(IV) intermediate (Compound I) and a tyrosine radical (Tyr °). The coproheme-bound ChdC structure revealed a helical-loop that is flexible and moves in towards the active site in the presence of substrate. This loop is hypothesized to act as an 'active site gate' which mediates substrate entry and product egress. Due to the cytotoxicity of heme and its porphyrin precursors, we proposed that the metabolite flux in this pathway is controlled by transient protein-protein interactions. Using the UV-Vis characteristics of porphyrins and phenotype characterization of the deltachdC knock-out strain of S. aureus complemented with ChdC point mutants, we present preliminary evidence for an interaction between ChdC the preceding enzyme of the pathway, CpfC. The same approaches also implicated potential interactions between ChdC and an unidentified heme-chaperone, which delivers heme to its final cellular destination. We propose that this chaperone is HemW. Experiments to test this hypothesis are outlined. This work elucidates yet different way that nature has equipped cells to perform radical chemistry in order to accomplish essential molecule transformations, such as that of decarboxylation and the simultaneous generation of CO 2, and emphasizes the importance of substrate/product post-catalysis cellular trafficking.Item Binding and repair of DNA by spore photoproduct lyase(Montana State University - Bozeman, College of Letters & Science, 2010) Zilinskas, Egidijus; Chairperson, Graduate Committee: Joan B. BroderickBacterial spores are extremely resistant to chemical and physical stresses, including UV irradiation, which in spores results in the formation of 5-thyminyl-5,6-dihydrothymine (spore photoproduct, SP). While SP accumulates in UV-irradiated bacterial spores, it is rapidly repaired during germination. Spore photoproduct lyase (SPL) is the enzyme that catalyzes the specific repair of spore photoproduct to two thymines. It utilizes S-adenosylmethionine (SAM) and a [4Fe-4S] cluster to catalyze this reaction, and is a member of the radical SAM superfamily. Presented here is an investigation of SPL repair activity towards stereochemically-defined synthetic R- and Sspore photoproduct dinucleosides and dinucleotides (SP and SPTpT, respectively), utilizing SPL purified from Clostridium acetobutylicum. The results of HPLC and Mass Spectrometry analysis of in vitro enzymatic assays demonstrate that SPL specifically repairs the 5R-, but not the 5S- isomer. The repair rates were determined to be ~0.4 nmol/min/mg of SPL for the 5R-SP dinucleoside and ~7.1 nmol/min/mg of SPL for the 5R-SPTpT dinucleotide. Since SPL binding to DNA is a key step in UV damage repair, SPL binding to undamaged DNA, as well as the 5R- and the 5S-isomers of SP and SPTpT, was also investigated. The binding to different substrates was investigated by carrying out electrophoretic mobility shift assays (EMSA) and time-resolved fluorescence decay experiments. SPL from both Bacillus subtilis and Clostridium acetobutylicum cooperatively binds the undamaged DNA with relatively high affinity (Kd = 4.7 x 10 -9 M for B.s. SPL and Kd = 1.7 x 10 -7 M for C.a. SPL). The presence of small, acid-soluble proteins (SASP), SAM or the [4Fe-4S] cluster of SPL have little effect on SPL binding to undamaged DNA. Interestingly, SPL is able to bind both the 5R- and the 5S- diastereomers of the synthetic dinucleoside/dinucleotide spore photoproduct, although only the 5R-isomer is repaired. SP lyase binding is stronger to the SPTpT dinucleotide than to the SP dinucleoside, likely due to the dinucleotide more closely resembling the natural substrate in double helical DNA. Also, SPL exhibits higher affinity towards SP and SPTpT than the repair products, thymidine or thymidylyl (3'-5') thymidine (TpT), respectively.Item Kinetic, mechanistic and spectroscopic studies of spore photoproduct lyase(Montana State University - Bozeman, College of Letters & Science, 2010) Silver, Sunshine Christine; Chairperson, Graduate Committee: Joan B. Broderick; Tilak Chandra, Egidijus Zilinskas, Eric M. Shepard, William E. Broderick and Joan B. Broderick were co-authors of the article, 'Spore photoproduct lyase catalyzes specific repair of the 5R but not the 5S spore photoproduct' in the journal 'Journal of the American chemical society' which is contained within this thesis.; Tilak Chandra, Egidijus Zilinskas, Shourjo Ghose, William E. Broderick, and Joan B. Broderick were co-authors of the article, 'Complete stereospecific repair of a synthetic dinucleotide spore photoproduct by spore photoproduct lyase' in the journal 'Journal of biological inorganic chemistry' which is contained within this thesis.; Shourjo Ghose, Jeffrey M. Buis and Joan B. Broderick were co-authors of the article, 'Kinetic and mechanistic insights into the repair of the spore photoproduct catalyzed by spore photoproduct lyase' in the journal 'Biochemistry' which is contained within this thesis.; David J. Gardenghi, BoiHanh Huynh, Robert K. Szilagyi and Joan B. Broderick were co-authors of the article, 'Combined Mössbauer and multi-edge x-ray absorption spectroscopic study of the Fe-S cluster in spore photoproduct lyase' in the journal 'Biochemistry' which is contained within this thesis.Spore forming organisms are a health threat to humans and other animals in part due to a remarkable resistance to UV irradiation. This resistance results from two events: first, the formation of a unique thymine dimer, 5-thyminyl-5,6-dihydrothymine (spore photoproduct, or SP) upon UV irradiation; and more importantly, the rapid and specific repair of this DNA photoproduct to two thymines by spore photoproduct lyase (SP lyase). Understanding the molecular basis of this radical-mediated DNA repair will ultimately allow for a better understanding of how to address the health risks caused by spore forming bacteria. SP lyase requires S-adenosyl-L-methionine and a [4Fe-4S] ¹+/²+ cluster to perform its catalysis. Presented in this work is a characterization of Clostridium acetobutylicm SP lyase and its ability to repair stereochemically defined dinucleoside and dinucleotide synthetic substrates. Careful synthesis and characterization followed by assays monitored by HPLC indicate SP lyase repairs only the 5R isomer of SP with an activity of 0.4 nmol/min/mg (dinucleoside substrate) and 7.1 nmol/min/mg (dinucleotide substrate). These results support the longstanding theory of SP formation by dimerization of adjacent thymines in double-helical DNA. Kinetic and mechanistic studies were pursued to further elucidate the mechanism of SP repair. Upon pre-reducing SP lyase, the specific activity increased nearly 4-fold to 1.29 umol/min/mg. Mechanistic studies utilizing [C-6- ³ H] SP DNA as the substrate revealed a primary tritium kinetic isotope effect of 16.1, indicating a rate determining step during the repair reaction. These results suggest nonstereospecific SP formation regarding the C-6 position and subsequent stereospecific abstraction of the C-6 H atom by SP lyase. Mössbauer and Fe/S K-edge X-ray absorption studies of anaerobically prepared SP lyase aided in further characterization of the [4Fe-4S] cluster and its interaction with SAM. The Fe K-edge EXAFS provide evidence for a slight cluster distortion upon interacting with SAM as a new spectral feature indicative of longer Fe-Fe distances is observed. The Fe K-edge XANES provide further support that SAM is not undergoing reductive cleavage in the presence of reduced SP lyase. Our XAS studies may provide new insights into the mechanism by which radical SAM enzymes initiate their diverse catalysis.