Structural and Functional Studies to Investigate Enzymatic Catalysis and Flavin Transfer Mechanism of The Two-component Alkanesulfonate Monooxygenase System from Escherichia coli

Abstract

Several bacterial organisms rely on the two-component alkanesulfonate monooxygenase system for the acquisition of organosulfonate compounds when inorganic sulfur is limiting in the environment. This system is comprised of an FMN reductase (SsuE) that supplies reduced flavin to the alkanesulfonate monooxygenase (SsuD). Desulfonation of alkanesulfonates by SsuD is catalyzed through the activation of dioxygen by reduced flavin. The three-dimensional structure of SsuD exists as a TIM-barrel fold with several discrete insertion regions. An extensive insertion region near the putative active site was unresolved in the SsuD structure, suggesting the importance of protein dynamics in the desulfonation mechanism. Three variants containing a partial deletion of the loop region were constructed to evaluate the functional properties of this region. There were no overall gross changes in secondary structure for the three SsuD deletion variants compared to wild-type SsuD, but each variant was found to be catalytically inactive. The deletion variants were unable to undergo the conformational changes necessary for catalysis even though they were able to bind reduced flavin. Results from rapid kinetic analyses suggested that the SsuD deletion variants failed to protect reduced flavin from non-enzymatic oxidation. These studies define the importance of the unresolved loop region for the protection of reduced flavin. A distinct feature of the alkanesulfonate monooxygenase system is that the flavin is utilized as a substrate instead of a bound prosthetic group. The reduced flavin is transferred from SsuE to SsuD, either through a dissociative or a channeling mechanism. In order to kinetically discern between a dissociative and a channeling flavin transfer mechanism, a method was designed using an SsuD variant with reduced flavin affinity to compete with wild-type SsuD for reduced flavin. Results from this novel competition method suggested that the flavin is transferred within a transient SsuE-SsuD complex during catalysis. In addition, the reductive half-reaction catalyzed by SsuE is affected by SsuD and its substrate under pre-steady-state kinetic conditions. The evidence provided in this study support a channeling flavin transfer mechanism in the two-component alkanesulfonate monooxygenase system.