Mechanistic Strategies of Two-component Flavin-dependent Monooxygenases Involved in Sulfur Acquisition
Author
Somai, Shruti
Abstract
All organisms require sulfur for diverse metabolic and physiological processes. Unlike mammals, bacteria are often deprived of sulfur in their natural habitat. To meet their sulfur needs, bacteria express various proteins that enable these microorganisms to utilize alternative sulfur sources during sulfur starvation. Some of the proteins expressed by bacteria are part of two-component flavin-dependent enzyme systems and include the alkanesulfonate monooxygenase (SsuE/SsuD), methanesulfinate monooxygenase (MsuE/MsuC), and methanesulfonate monooxygenase (MsuE/MsuD) systems. These two-component flavin-dependent systems consist of a flavin reductase (SsuE and MsuE) that supplies reduced flavin to the monooxygenase (SsuD, MsuC, MsuD) for the coordinated desulfonation of alkanesulfonates to obtain sulfite which is ultimately used in the synthesis of sulfur-containing biomolecules.
Of specific interest in this dissertation are the TIM-barrel enzymes, SsuD and MsuD, that share 65% amino acid sequence identity. A combination of kinetic and computational analyses was performed to evaluate why these structurally and functionally similar monooxygenases had different substrate specificities. SsuD was unable to utilize methanesulfonate and ethanesulfonate as a sulfur substrate in desulfonation assays, even though both SsuD and MsuD had similar binding affinities for reduced flavin and methanesulfonate. Additionally, MsuD showed decreased proteolytic susceptibility in the presence of FMNH2 and methanesulfonate as compared to the SsuD/FMNH2/methanesulfonate and SsuD/FMNH2/octanesulfonate complexes. Structural analysis using accelerated molecular dynamic simulations revealed that methanesulfonate is not stabilized within the active site of SsuD due to the absence of a longer alkyl chain that provides an appropriate active site arrangement for catalysis. Under physiological conditions, SsuD would require molecular oxygen to successfully desulfonate alkanesulfonates and yield sulfite. Even though results from fluorometric titration experiments revealed that SsuD was able to bind methanesulfonate, this might not represent a catalytically active complex given that the experiments were performed anaerobically.
The two-component alkanesulfonate monooxygenase system (SsuE/SsuD) is commonly found in diverse bacteria highlighting its importance in adequately providing bacteria with alternative sulfur sources. The mechanism of this enzyme system is of extreme importance in bacterial sulfur acquisition; however, mechanistic details remain elusive. While SsuD has previously been proposed to employ a C4a-(hydro)peroxy flavin as an oxygenating flavin intermediate, a flavin-N5-oxide was identified as an oxygenating intermediate or formed as the final product during catalytic turnover by several bacterial flavin-dependent enzymes. Structural similarity with some of these enzymes led to the hypothesis that SsuD could also employ a flavin-N5-oxide as an oxygenating intermediate or as the final product. Studies presented in this dissertation were focused on determining the final flavin product in reaction of SsuD. The results obtained revealed that SsuD does not form the flavin-N5-oxide as a final product, thereby suggesting that this flavin adduct could be utilized as one of the oxygenating flavin intermediates in the reaction catalyzed by SsuD. Alternatively, a flavin-N5-oxide might not be formed by SsuD, and instead an N5-peroxy flavin intermediate could be employed to perform the oxidative half-reaction. Moreover, the studies presented in this dissertation focused on elucidating the roles conserved residues and structural features play in oxygen activation to perform the desulfonation reaction catalyzed by SsuD.
While the alkanesulfonate sulfonate monooxygenase system is commonly found in bacteria, certain bacteria including Pseudomonas species utilize a more complex enzyme system to acquire sulfur during sulfur starvation. This includes the methanesulfinate (MsuE/MsuC) and the methanesulfonate (MsuE/MsuD) monooxygenase systems. Unlike SsuD and MsuD, MsuC is structurally distinct and relies on MsuE to supply reduced flavin for the oxidation of methanesulfinate to yield methanesulfonate. The studies presented in this dissertation are the first investigating the structural and mechanistic features of MsuC from Pseudomonas aeruginosa.
MsuC was shown to have a higher affinity for the reduced flavin form and preferred FMNH2 in fluorometric titration experiments. While flavoenzymes have been reported to undergo changes in the oligomeric state in the presence of substrates and products to efficiently transfer reduced flavin, the presence of substrates and products did not alter the structural and thermal stability of MsuC. Additionally, rapid reaction kinetic experiments evaluating the reduced flavin transfer between MsuE and MsuC revealed that reduced flavin was protected by MsuC in the absence of methanesulfinate. This enzymatic protection prevents the autooxidation of reduced flavin which could subsequently result in the generation of reactive oxygen species. Finally, the viability of different transposon mutants was tested in the presence of various sulfur sources in order to evaluate their role within the cell. Interestingly, the MsuC transposon mutant was able to utilize methanesulfinate as a sulfur source, thereby raising questions regarding its previously assigned role as a methanesulfinate monooxygenase.
Taken together, the findings presented in this dissertation provide structural and mechanistic insight into the roles diverse flavin-dependent monooxygenases fulfill during sulfur starvation. Given the fact that these enzymes are also found in a wide range of pathogenic bacteria such as the multi-drug resistant Pseudomonas aeruginosa, which is one of the leading causes of nosocomial infections, the structural and mechanistic information obtained from these studies could represent a notable target for rational drug design.
Date
2023-08-09
Citation:
APA:
Somai, Shruti.
(August 2023).
Mechanistic Strategies of Two-component Flavin-dependent Monooxygenases Involved in Sulfur Acquisition
(Doctoral Dissertation, East Carolina University). Retrieved from the Scholarship.
(http://hdl.handle.net/10342/13144.)
MLA:
Somai, Shruti.
Mechanistic Strategies of Two-component Flavin-dependent Monooxygenases Involved in Sulfur Acquisition.
Doctoral Dissertation. East Carolina University,
August 2023. The Scholarship.
http://hdl.handle.net/10342/13144.
April 28, 2024.
Chicago:
Somai, Shruti,
“Mechanistic Strategies of Two-component Flavin-dependent Monooxygenases Involved in Sulfur Acquisition”
(Doctoral Dissertation., East Carolina University,
August 2023).
AMA:
Somai, Shruti.
Mechanistic Strategies of Two-component Flavin-dependent Monooxygenases Involved in Sulfur Acquisition
[Doctoral Dissertation]. Greenville, NC: East Carolina University;
August 2023.
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Publisher
East Carolina University