THE π-H ELIX IN FMN-DEPENDENT REDUCTASES PROMOTES OLIGOMERIC CHANGES TO COORDINATE THE MECHANISMS OF THE ALKANESULFONATE MONOOXYGENASE SYSTEMS
Author
Aloh, Chioma Helen
Abstract
Bacteria two-component flavin-dependent systems are upregulated for sulfur acquisition under sulfur limiting conditions. Two-component flavin-dependent systems are comprised of a reductase (SsuE/MsuE) that catalyzes the reduction of flavin, and the monooxygenase (SsuD/MsuD) catalyzes the desulfonation of organosulfur compounds for sulfur acquisition. The unique reactions catalyzed by these systems are enabled by a diverse range of structural features: π-helix, oligomeric changes, and protein-protein interactions. However, it is not clear how these structural features work synergistically to catalyze flavin reduction, transfer, and desulfonation. In this study, we take a holistic view of these structural features by investigating how they are coordinated for overall catalysis.
SsuE and MsuE belong to the NADPH:FMN reductase family and they share 30% amino acid identity. Results from several studies revealed that SsuE exists in different oligomeric states depending on the technique utilized. However, the precise oligomeric state of the reductases and how it functions in flavin reduction and transfer have not been explored. The oligomeric state of SsuE and MsuE were determined in the presence and absence of substrates. SsuE exists as a tetramer but shifts to a tetramer-dimer equilibrium in the presence of NADPH or FMN. Interestingly, MsuE exists as a dimer in the apo form and with NADPH present. However, MsuE shifts from a dimer to a tetramer in the presence of flavin. These results reveal that the oligomeric state of SsuE and MsuE is altered in the presence of substrates. Transfer of reduced flavin from SsuE to SsuD was observed to occur through protein-protein interactions. Experiments were performed to determine if the oligomeric changes of SsuE are coordinated with the formation of protein-protein interactions with SsuD. In the presence of FMN, the proposed SsuE-SsuD complex was formed and the dimeric form of SsuE was the predominant form. The dimer interface of SsuE will expose protein interaction sites required for the efficient transfer of reduced flavin. The dimer-dimer interface of SsuE houses the π-helix which has been proposed to be responsible for flavin transfer. The π-helix is a distinct secondary structure which is characterized by an insertional residue into an α-helix that differentiates canonical flavoproteins from two-component FMN reductases. Results from previous studies revealed that the insertional residue is not solely responsible for the structural differences between these reductases. The insertional residue in SsuE is the Tyr128 and a Tyr126 was found in a similar position in a canonical flavoprotein, chromate reductase (ChrR). Clearly, additional structural adaptations may be responsible for the structural divergence between these reductases. Variants of ChrR (Q132P, Q127D, Q132P/127D ChrR) were generated from the conserved residues on the π-helical region of two-component FMN reductases. There was no measurable oxidase activity nor desulfonation activity with SsuD for any of the ChrR variants. Interestingly, wild-type and Q132P ChrR were capable of transferring electrons to ferricyanide in ferricyanide assays. However, Q127D and Q132P/127D ChrR had no measurable ferricyanide activity. Further studies would need to be performed to evaluate the structural differences between these reductases. The unique π-helix of FMN reductases clearly bridges the functionality of the oligomeric changes of SsuE that are linked to protein-protein interactions critical for reduced flavin transfer.
Date
2023-08-08
Citation:
APA:
Aloh, Chioma Helen.
(August 2023).
THE π-H ELIX IN FMN-DEPENDENT REDUCTASES PROMOTES OLIGOMERIC CHANGES TO COORDINATE THE MECHANISMS OF THE ALKANESULFONATE MONOOXYGENASE SYSTEMS
(Doctoral Dissertation, East Carolina University). Retrieved from the Scholarship.
(http://hdl.handle.net/10342/13145.)
MLA:
Aloh, Chioma Helen.
THE π-H ELIX IN FMN-DEPENDENT REDUCTASES PROMOTES OLIGOMERIC CHANGES TO COORDINATE THE MECHANISMS OF THE ALKANESULFONATE MONOOXYGENASE SYSTEMS.
Doctoral Dissertation. East Carolina University,
August 2023. The Scholarship.
http://hdl.handle.net/10342/13145.
May 04, 2024.
Chicago:
Aloh, Chioma Helen,
“THE π-H ELIX IN FMN-DEPENDENT REDUCTASES PROMOTES OLIGOMERIC CHANGES TO COORDINATE THE MECHANISMS OF THE ALKANESULFONATE MONOOXYGENASE SYSTEMS”
(Doctoral Dissertation., East Carolina University,
August 2023).
AMA:
Aloh, Chioma Helen.
THE π-H ELIX IN FMN-DEPENDENT REDUCTASES PROMOTES OLIGOMERIC CHANGES TO COORDINATE THE MECHANISMS OF THE ALKANESULFONATE MONOOXYGENASE SYSTEMS
[Doctoral Dissertation]. Greenville, NC: East Carolina University;
August 2023.
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Publisher
East Carolina University