Probing the flavin transfer mechanism in alkanesulfonate monooxygenase system

Bacteria acquire sulfur through the sulfur assimilation pathway, but under sulfur limiting conditions bacteria must acquire sulfur from alternative sources. The alkanesulfonate monooxygenase enzymes are expressed under sulfur-limiting conditions, and catalyze the desulfonation of wide-range of alkanesulfonate substrates. The SsuE enzyme is an NADPH-dependent FMN reductase that provides reduced flavin to the SsuD monooxygenase. The mechanism for the transfer of reduced flavin in flavin dependent two-component systems occurs either by free-diffusion or channeling. Previous studies have shown the presence of protein-protein interactions between SsuE and SsuD, but the identification of putative interaction sights have not been investigated. Current studies utilized HDX-MS to identify protective sites on SsuE and SsuD. A conserved α-helix on SsuD showed a decrease in percent deuteration when SsuE was included in the reaction. This suggests the role of α-helix in promoting protein-protein interactions. Specific SsuD variants were generated in order to investigate the role of these residues in protein-protein interactions and catalysis. Variant containing substitutions at the charged residues showed a six-fold decrease in the activity, while a deletion variant of SsuD lacking the α-helix showed no activity when compared to wild-type SsuD. In addition, there was no protein-protein interactions identified between SsuE and his-tagged SsuD variants in pull-down assays, which correlated with an increase in the Kd value. The α-helix is located right next to a dynamic loop region, positioned at the entrance of the active site. The putative interaction site and dynamic loop region located so close to the active site of SsuD suggests the importance of this region in the SsuD catalysis. Stopped-flow studies were performed to analyze the lag-phase which signifies the stabilization and transfer of reduced flavin from SsuE to SsuD. The SsuD variants showed a decrease in lag-phase, which could be because of a downturn in flavin transfer. A competitive assay was devised to evaluate the mechanism of flavin transfer in the alkanesulfonate monooxygenase system. A variant of SsuE was generated which interacted with SsuD, but was not able to reduce FMN. Assays that included varying concentrations of Y118A SsuE and wild-type SsuE in the coupled assays showed a decrease in the desulfonation activity of SsuD. The decrease in activity could be by virtue of Y118A SsuE competing with the wild-type SsuE for the putative docking site on SsuD. These studies define the importance of protein-protein interactions for the efficient transfer of reduced flavin from SsuE to SsuD leading to the desulfonation of alkanesulfonates.


Introduction
Sulfur is essential for the survival and growth of all living organisms. Bacteria utilize inorganic sulfur for the biosynthesis of sulfur-containing amino acids and cofactors. Sulfur exists as sulfonate and sulfonate esters in the soil. These xenobiotic compounds are hard to break down and require enzymatic activity for their catalysis. Therefore bacteria must have an alternative mechanism to acquire sulfur when sulfur is limiting (1). Bacteria express a specific set of proteins under sulfur-limitation conditions (2). The sulfonate-sulfur utilization (ssu) proteins enable bacteria to utilize xenobiotic compounds like alkanesulfonates as a sulfur source. The ssu operon is induced when sulfur is limiting and encodes a NADPHdependent FMN-reductase (SsuE) and FMNH 2 dependent alkanesulfonate monooxygenase (SsuD) (2,3). SsuE reduces FMN to FMNH 2 which is then transferred to SsuD. The SsuD enzyme utilizes the reduced flavin to catalyze the oxygenolytic cleavage of the C-S bond in alkanesulfonate (4). Depending on the system being studied, the transfer of reduced flavin can occur either by a channeling or dissociative mechanism. For some flavin dependent twocomponent systems the transfer of reduced flavin can be best explained as a combination of the two mechanisms (5). The flavin transfer mechanism in the flavin-dependent alkanesulfonate monooxygenase system is important to understand the catalytic mechanism of this two-component system.

Previous studies have reported the presence of physical interactions between SsuE and
SsuD leading to the formation of a transient complex (6). Recent studies have utilized HDX-MS to highlight specific regions on SsuE and SsuD involved in protein-protein interactions (7). These regions were protected from hydrogen-deuterium exchange due to protein-protein interactions between SsuE and SsuD. Notable charged amino acid residues were reported on the protected regions of SsuD that were shown to be involved in protein-protein interactions.
Substitutions of these protected regions had a direct impact on SsuD activity, highlighting the role of protein-protein interactions in the transfer of reduced flavin. Previous studies evaluating protein-protein interactions and substrate binding suggest that a cooperative mechanism is involved in the transfer of reduced flavin between SsuE to SsuD. The studies described will help to enhance knowledge regarding reduced flavin transfer in twocomponent systems. Previous studies have already highlighted the regions involved in protein-protein interactions between SsuE and SsuD. To identify the role of protein-protein interactions an inactive SsuE variant was used in a competitive assay with wild-type SsuD and SsuE. Also the rate of production of reactive oxygen species, due to the autoxidation of reduced flavin, was calculated using resazurin dye. Single turnover stopped-flow kinetics was performed to determine the stability of reduced flavin upon substitutions at the interaction sites. The reported results will enable us to understand the mechanism of reduced flavin transfer in the two-component alkanesulfonate monooxygenase system.

Materials
All chemicals were purchased from SigmaAldrich, BioRad, or Fisher. Escherichia coli strain BL21(DE3) was purchased from Stratagene (La Jolla, CA). DNA primers were synthesized by Invitrogen (Carlsbad, CA). The expression and purification of wildtype SsuE and SsuD variants was performed as previously reported (10).

Construction, expression and purification of recombinant proteins
Previous studies have identified protected peptides on SsuE and SsuD by HDX-MS (7).
The charged amino acids were substituted and deleted to generate DDE (251/252/253) AAA and ∆D251-A261 SsuD variants. The Tyr118 of SsuE was also substituted to generate an inactive variant Y118A SsuE. The substitutions and deletions of amino acids were performed as described (7). The expression and purification of all SsuD variants was performed as previously reported (12).

Kinetics of resazurin reduction
Stopped-flow kinetic analyses were performed to evaluate reactive oxygen species upon autoxidation of FMNH 2 due to unsuccessful transfer to SsuD variants

Where, [A] o is the initial concentration of Resazurin dye, [A] is the final concentration of
Resazurin dye, and k represents the rate of reduction of Resazurin at 570 nm (k 570 ).

Competition assay
Spectrofluorimetric analyses were performed to determine the binding affinity of Y118A SsuE with SsuD (6). The protein samples were excited at 450 nm and emission intensity measurements were at 524 nm. Aliquots of SsuD (0.02 -0.95 µM) were titrated against FMN-bound SsuE. Similar experiments were performed for wild-type SsuE. The equation used to determine the concentration of SsuD bound to SsuE was (7): Where, [SsuE] represents the initial concentration of enzyme, I 0 is the initial fluorescence intensity of FMN prior to addition of SsuD, I c is the fluorescence intensity of FMN following each addition of SsuD, and I f is the final fluorescent intensity. The concentration of [SsuD] bound (y) were then separately plotted against [FMN] total and [SsuD] total (x) respectively, to obtain the dissociation constant (K D ) for SsuE and SsuD binding by using following equation (): The linear dependence of SsuD activity on the concentration of SsuE was first established.

Rapid Reaction Kinetic studies of Flavin Transfer
The role of protein-protein interaction in the transfer of reduced flavin from ssue to the SsuD variants was probed through rapid reaction kinetic analyses. This experiment was performed to evaluate if reduced flavin has decreased stability due to a decrease in protein-protein

Binding and Competitive analyses of Y118A SsuE
If

Discussion
The flavin-dependent alkanesulfonate monooxygenase system is a two- the two enzymes (15)(16)(17)(18)(19)(20)(21)(22). Previously studied flavin-dependent two-component systems propose a direct or dissociative mechanism for the transfer of reduced flavin. A dissociative mechanism for the transfer of reduced flavin is prevalent in many two-component systems (23,24,25). The dissociative mechanism depends on passive diffusion of reduced flavin for its transfer from the reductase to the monooxygenase. In the flavin-dependent two-component enzyme systems the flavin reductases have a higher affinity for oxidized flavin and the monooxygenases have a higher affinity for reduced flavin (8,25,26,27,28). The dissociative mechanism is based on the rapid transfer of flavin between the reductase and monooxygenase half of enzymes. Conversely, a direct transfer mechanism minimizes the contact between reduced flavin and the external environment (6,29,30). The direct transfer mechanism enhances the transfer of reduced flavin by either forming a molecular channel or by bridging the distance between two-active sites by bringing them in close proximity to each other through protein-protein interactions. Various kinetic and biophysical studies have provided support for a channeling mechanism for the transfer of reduced flavin between FRP and bacterial luciferase (29,30). Steady-state kinetic studies on styrene monooxygenase (SMO) have shown the presence of both a dissociative and direct transfer mechanism for reduced flavin transfer (31). The presence of site-specific protein-protein interactions between SsuE and SsuD have been suggested in the alkanesulfonate monooxygenase system (6,7). The HDX-MS experiment identified protected regions on SsuE and SsuD, which were shown to play a key role in protein-protein interactions (7). The protected region of SsuD had a highly conserved α-helix, which was located adjacent to the active site. The α-helix of SsuD appeared to play a key role in dynamic and conformational changes required for interaction with SsuE. The protein-protein interactions promoted by the movement of the conserved αhelix will bring the two active sites in close proximity and increase the effectiveness of reduced flavin transfer between SsuE and SsuD.
In  (7). The disruption at this helical region affected not only the binding affinity of SsuD towards SsuE but also the efficiency of reduced flavin transfer. The alkanesulfonate monooxygenase system is a stress-response protein that gets activated during sulfate-limiting conditions. Thus the regulation of these enzymes is tightly regulated with specific functions in order to replenish bacteria with sulfate.