Date of Award
8-2024
Document Type
Campus Access Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry/Biological Chemistry
First Advisor
Daniel P. Dowling
Second Advisor
Jason Evans
Third Advisor
Jennifer Bridwell-Rabb, Niya Sa
Abstract
Sulfur is essential for life, playing a critical role in metabolic functions and cellular signaling pathways. While bacteria typically obtain sulfur from inorganic sulfate, they must resort to alternative sources when sulfate is limited. Pseudomonads have evolved a sophisticated system to address this challenge, employing two-component flavin-dependent monooxygenases (TC-FMOs) to assimilate sulfur from alkanesulfonates and dialkylsulfones, such as dimethylsulfone (DMSO2). This process is triggered by a stress response during sulfur starvation conditions. To convert DMSO2 into bioavailable sulfite, Pseudomonads perform a series of chemical transformations regulated by the msu and sfn operons. This dissertation investigates the mechanisms underlying these transformations in Pseudomonas fluorescens, focusing on the TC-FMOs responsible for DMSO2 catabolism. The insights gained from this research have potential applications in bioremediation and may contribute to our understanding of human health in relation to other Pseudomonas species. The msu and sfn operons encode the two-component flavin-dependent monooxygenases (TC-FMOs) SfnG, MsuC, and MsuD, along with the FMN reductase MsuE, which collectively catalyzes the catabolism of dimethylsulfone (DMSO2) to sulfite. This dissertation provides an in depth structural and biophysical investigation into the function of SfnG and MsuC, which convert DMSO2 to methanesulfinate (MSI–) and MSI– to methanesulfonate (MS–), respectively. Multiple high-resolution crystal structures of SfnG revealed critical protein regions that become ordered upon binding of the ligands FMN and DMSO2. The placement of ligands identified a putative oxygen binding site, suggesting SfnG may employ an N5-(hydro)peroxyflavin for oxidative cleavage of the C–S bond of DMSO2. Functional biochemical experiments support the use of an N5-(hydro)peroxyflavin intermediate, and biophysical studies reveal the complexities of ligand binding to SfnG. The second enzyme in the pathway, MsuC, catalyzes the oxidation of MSI– to form MS– through S–O bond formation. Structural analysis confirms that MsuC belongs to the two-component flavin-dependent monooxygenase (TC-FMO) family and likely employs C4a flavin chemistry for catalysis. A high resolution unliganded structure, combined with computational studies, revealed a putative MSI– binding pocket with features that allow for binding of this negatively charged molecule. Further structural analysis, including alignments with homologous enzymes, uncovered conserved C-terminal interactions that stabilize the active site and are connected to substrate binding. These structural inferences provide a foundation for future functional studies, which will enhance our understanding of MsuC’s catalytic mechanism and substrate binding dynamics. Additionally, biophysical characterization has yielded new insights into the role of FMN binding in enzyme stability and function. The high-resolution structures of SfnG with bound FMN and DMSO2, as well as MsuC, have provided valuable insights into these enzymes. Detailed characterization of their structures and active sites enables targeted mutational analyses to elucidate substrate specificity and promiscuity. Notably, these systems are prevalent in various Pseudomonas strains, including opportunistic, multi-drug resistant pathogens like Pseudomonas aeruginosa. Given that SfnG and MsuC catalyze C–S bond cleavage and S–O bond formation, respectively, future studies could explore modulating their activities to target other compounds, such as those containing C–Si bonds or persistent environmental contaminants like polyfluoroalkyl substances (PFAS). This research not only advances our understanding of bacterial sulfur metabolism but also opens avenues for potential biotechnological and environmental applications.
Recommended Citation
Gonzalez, Reyaz, "Insights into the Sulfur Assimilation Pathway of Dimethylsulfone During Sulfur Starvation in Pseudomonas fluorescens: Structural and Biophysical Analysis of Two-Component Flavin-Dependent Monooxygenases SfnG and MsuC" (2024). Graduate Doctoral Dissertations. 997.
https://scholarworks.umb.edu/doctoral_dissertations/997
Comments
Free and open access to this Campus Access Thesis is made available to the UMass Boston community by ScholarWorks at UMass Boston. Those not on campus and those without a UMass Boston campus username and password may gain access to this thesis through resources like Proquest Dissertations & Theses Global (https://www.proquest.com/) or through Interlibrary Loan. If you have a UMass Boston campus username and password and would like to download this work from off-campus, click on the "Off-Campus UMass Boston Users