Alkanesulfonate Monooxygenase System Research Articles

Investigating Structural Features Utilized by Two‐component Flavin‐dependent Systems

Many enzymes exist in different oligomeric states which serves to regulate enzyme activity, integrate different pathways, expose new active sites at the interface of the interacting subunit, and enhance protein stability. Two‐component FMN‐dependent systems expressed in bacteria during times of limited sulfur availability regulate enzyme activity through the coordination of oligomeric changes of the reductase with reduced flavin transfer to the monooxygenase. The alkanesulfonate monooxygenase system is ubiquitous in bacteria, the reductase (SsuE) and the monooxygenase (SsuD) catalyze the desulfonation of alkanesulfonates to the corresponding aldehyde and sulfite. Pseudomonas aeruginosa has a more complex mechanism for acquiring sulfur. In addition to the SsuE/SsuD system, the organism also encodes a flavin‐dependent reductase (MsuE) and two monooxygenases (MsuC/MsuD) that together convert methanesulfinate to formaldehyde and sulfite. In the alkanesulfonate monooxygenase systems, the FMN reductases exist in different oligomeric states. SsuE exists as a tetramer (dimer of dimers) but can shift to a dimer in the presence of flavin and/or substrate. Coordinated structural dynamics of the FMN reductases and monooxygenase enables efficient synchronization of labile flavin transfer and catalysis. However, the unique structural features that integrates the oligomeric state of the reductase with the mode of flavin transfer to the monooxygenase has not been fully explored.Protein‐protein interactions between the FMN reductases and monooxygenases prevent the unproductive oxidation of reduced flavin. Studies were performed to investigate the interaction sites and oligomeric alterations critical for the transfer of reduced flavin from the reductase to the partner monooxygenase. SsuE and MsuE share 30% amino acid identity and SsuD and MsuD have 60% amino acid sequence identity, implying that although the enzymes utilize different substrates, they share similar structural strategies to regulate flavin transfer. Results from fluorescence spectroscopy show a high binding affinity between SsuE/SsuD and MsuE/MsuD. The high binding affinity observed ensures the safe transfer of reduced flavin from the reductase to the monooxygenase. Protein‐protein interactions sites were identified using hydrogen‐deuterium exchange with mass spectrometry (HDX‐MS) to evaluate flavin transfer in the alkanesulfonate monooxygenase systems (SsuE/SsuD and MsuE/MsuD). The interaction sites in SsuE are located at the dimer‐dimer interface and are close to the active site, while the monooxygenase interactions sites are conserved across SsuD enzymes from different bacterial sources. Comparable protein‐protein interaction sites observed in the alkanesulfonate monooxygenase system (SsuE/SsuD) were identified in the methanesulfinate monooxygenase system (MsuE/MsuD). Taken together, oligomeric changes of the flavin reductase regulates the transfer of flavin and the dimer‐dimer interface houses the sites for protein‐protein interactions with the monooxygenase facilitating catalysis.

Not as easy as π: An insertional residue does not explain the π-helix gain-of-function in two-component FMN reductases.

The π-helix located at the tetramer interface of two-component FMN-dependent reductases contributes to the structural divergence from canonical FMN-bound reductases within the NADPH:FMN reductase family. The π-helix in the SsuE FMN-dependent reductase of the alkanesulfonate monooxygenase system has been proposed to be generated by the insertion of a Tyr residue in the conserved α4-helix. Variants of Tyr118 were generated, and their X-ray crystal structures determined, to evaluate how these alterations affect the structural integrity of the π-helix. The structure of the Y118A SsuE π-helix was converted to an α-helix, similar to the FMN-bound members of the NADPH:FMN reductase family. Although the π-helix was altered, the FMN binding region remained unchanged. Conversely, deletion of Tyr118 disrupted the secondary structural properties of the π-helix, generating a random coil region in the middle of helix 4. Both the Y118A and Δ118 SsuE SsuE variants crystallize as a dimer. The MsuE FMN reductase involved in the desulfonation of methanesulfonates is structurally similar to SsuE, but the π-helix contains a His insertional residue. Exchanging the π-helix insertional residue of each enzyme did not result in equivalent kinetic properties. Structure-based sequence analysis further demonstrated the presence of a similar Tyr residue in an FMN-bound reductase in the NADPH:FMN reductase family that is not sufficient to generate a π-helix. Results from the structural and functional studies of the FMN-dependent reductases suggest that the insertional residue alone is not solely responsible for generating the π-helix, and additional structural adaptions occur to provide the altered gain of function.

Functional Evaluation of the π-Helix in the NAD(P)H:FMN Reductase of the Alkanesulfonate Monooxygenase System.

A subgroup of enzymes in the NAD(P)H:FMN reductase family is comprised of flavin reductases from two-component monooxygenase systems. The diverging structural feature in these FMN reductases is a π-helix centrally located at the tetramer interface that is generated by the insertion of an amino acid in a conserved α4 helix. The Tyr insertional residue of SsuE makes specific contacts across the dimer interface that may assist in the altered mechanistic properties of this enzyme. The Y118F SsuE variant maintained the π-π stacking interactions at the tetramer interface and had kinetic parameters similar to those of wild-type SsuE. Substitution of the π-helical residue (Tyr118) to Ala or Ser transformed the enzymes into flavin-bound SsuE variants that could no longer support flavin reductase and desulfonation activities. These variants existed as dimers and could form protein-protein interactions with SsuD even though flavin transfer was not sustained. The ΔY118 SsuE variant was flavin-free as purified and did not undergo the tetramer to dimer oligomeric shift with the addition of flavin. The absence of desulfonation activity can be attributed to the inability of ΔY118 SsuE to promote flavin transfer and undergo the requisite oligomeric changes to support desulfonation. Results from these studies provide insights into the role of the SsuE π-helix in promoting flavin transfer and oligomeric changes that support protein-protein interactions with SsuD.

Transformation of a Flavin-Free FMN Reductase to a Canonical Flavoprotein through Modification of the π-Helix.

The flavin reductase of the alkanesulfonate monooxygenase system (SsuE) contains a conserved π-helix located at the tetramer interface that originates from the insertion of Tyr118 into helix α4 of SsuE. Although the presence of π-helices provides an evolutionary gain of function, the defined role of these discrete secondary structures remains largely unexplored. The Tyr118 residue that generated the π-helix in SsuE was substituted with Ala to evaluate the functional role of this distinctive structural feature. Interestingly, generation of the Y118A SsuE variant converted the typically flavin-free enzyme to a flavin-bound form. Mass spectrometric analysis of the extracted flavin gave a mass of 457.11 similar to that of the FMN cofactor, suggesting the Y118A SsuE variant retained flavin specificity. The Y118A SsuE FMN cofactor was reduced with approximately 1 equiv of NADPH in anaerobic titration experiments, and the flavin remained bound following reduction. Although reactivity of the reduced flavin with oxygen was slow in NADPH oxidase assays, the variant supported electron transfer to ferricyanide. In addition, there was no measurable sulfite product in coupled assays with the Y118A SsuE variant and SsuD, further demonstrating that flavin transfer was no longer supported. The results from these studies suggest that the π-helix enables SsuE to effectively utilize flavin as a substrate in the two-component monooxygenase system and provides a foundation for further studies aimed at evaluating the functional properties of the π-helix in SsuE and related two-component flavin reductase enzymes.

Structural Properties in the FMN Reductase of the Alkanesulfonate Monooxygenase System that Dictate Oligomeric States and Catalysis

The alkanesulfonate monooxygenase system is expressed in many bacteria when sulfur is limiting to enable the organism to utilize a diverse range of alkanesulfonates. This system is comprised of a NADPH‐dependent FMN reductase (SsuE) that reduces flavin and transfers it to the monooxygenase enzyme (SsuD) for the desulfonation of alkanesulfonates 1. The SsuE enzyme exists in different oligomeric states, but it is unclear how these oligomeric states relate to catalysis.A conserved π‐helix is located in the tetramer interface of SsuE which results from the insertion of Tyr118 in the α4‐helix. This Tyr118 residue forms a hydrogen bond with the carboxyl group of Ala78 across the tetramer interface to enhance the protein architecture 2. Variants of Y118 SsuE were generated to evaluate the role of the π‐helix in SsuE. Interestingly, the Y118A and Y118S SsuE variants were FMN‐bound as purified, while the Y118F and ΔY118 SsuE variants were flavin‐free. The Y118 SsuE variants were able to effectively bind FMN giving similar Kd values as wild‐type SsuE. This flavin affinity correlated with the ability of Y118 SsuE to reduce flavin with kcat/Km values similar to that of wild‐type SsuE in flavin reductase assays. While desulfonation activity was observed with the Y118F SsuE variant, there was no activity in the coupled assays monitoring SsuD desulfonation with the Y118A, Y118S and the ΔY118 variants of SsuE. This would indicate that the Tyr118 is vital for the transfer of FMNH2 to SsuD. The results from these studies suggest that multiple non‐covalent interactions are important for the structural stability of SsuE. These findings provide insights on the role of Tyr118 in SsuE.
 1. Eichhorn, E. et al. (1999) J. Biol. Chem. 274, 26639‐46.
 2. Driggers, C. M. et al (2014) Biochem. 53, 3509‐19.

Steady-State Kinetic Isotope Effects Support a Complex Role of Arg226 in the Proposed Desulfonation Mechanism of Alkanesulfonate Monooxygenase

The alkanesulfonate monooxygenase system catalyzes the desulfonation of alkanesulfonates through proposed acid-base mechanistic steps that involves the abstraction of a proton from the alkane peroxyflavin intermediate and protonation of the FMN-O(-) intermediate. Both solvent and kinetic isotope studies were performed to define the proton transfer steps involved in the SsuD reaction. Substitution of the protium at the C1 position of octanesulfonate with deuterium resulted in an observed primary isotope effect of 3.0 ± 0.2 on the kcat parameter, supporting abstraction of the α-proton from the alkane peroxyflavin as the rate-limiting step in catalysis. Previous studies implicated Arg226 as the acid involved in the reprotonation of the hydroxyflavin intermediate. Solvent isotope kinetic studies gave an inverse isotope effect on (D2O)kcat of 0.75 ± 0.04 with no observable effect on (D2O)kcat/Km. This resulted in equivalent solvent isotope effects on (D2O)kcat and (D2O)(kcat)D, suggesting a solvent equilibrium isotope effect on a step occurring after the first irreversible step through product release. Data from proton inventory studies on kcat were best fit to a dome-shaped curve consistent with a conformational change to an open conformation during product release. The solvent isotope effect data coupled with the corresponding proton inventory results support and extend our previous observations that Arg226 donates a proton to the FMN-O(-) intermediate, triggering a conformational change that opens the enzyme to solvation and promotes product release.

Identifying the Mechanism of Reduced Flavin Transfer in Alkanesulfonate Monooxygenase System

The two‐component alkanesulfonate monooxygenase system from bacteria are involved in the acquisition of sulfonates during sulfurlimiting conditions. In this two‐component system the FMN reductase (SsuE) provides reduced flavin to the monooxygenase enzyme (SsuD). The monooxygenase enzyme (SsuD) catalyzes the oxygenolytic cleavage of the carbon‐sulfur bond of alkanesulfonates, leading to the formation of the corresponding aldehyde and sulfite product.The transfer of reduced flavin from SsuE to SsuD may involve either a channeling or dissociative mechanism. Prior studies have identified stable protein‐protein interactions between SsuE and SsuD supporting a channeling mechanism for flavin transfer. The SsuD enzyme belongs to the bacterial luciferase family that includes bacterial luciferase and LadA. Members of this family have a characteristic TIM‐barrel conformation with several insertion regions. The mobile loop contained on the insertion region was shown to be involved in stabilizing loop closure to protect reaction flavin intermediates, but the role of this region in flavin transfer has not been evaluated. HDX‐MS studies were conducted to identify potential interaction sites on each enzyme. The SsuD protected sites were located on the mobile loop region, while the protected sites on SsuE were near the putative active site region. There are noticeable charged and polar residues in these regions, which may play a role in the formation of a transient stable‐structure. Variants of these polar regions were generated to evaluate protein‐protein interactions and flavin transfer were altered. The interaction sites on SsuE and SsuD have the potential to act as a dynamic region which enables the transfer of reduced flavin between the two proteins.

Deletional studies to investigate the functional role of a dynamic loop region of alkanesulfonate monooxygenase

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 disordered 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. Rapid kinetic analyses monitoring the reductive and oxidative half-reactions indicated that the SsuD deletion variants failed to protect reduced flavin from unproductive oxidation. These studies define the importance of dynamic loop region for protection and stabilization of reduced flavin and reaction intermediates.

Mechanism for sulfur acquisition by the alkanesulfonate monooxygenase system

The bacterial alkanesulfonate monooxygenase system is involved in the acquisition of sulfur from organosulfonated compounds during limiting sulfur conditions. The reaction relies on an FMN reductase to supply reduced flavin to the monooxygenase enzyme. The reaction catalyzed by the alkanesulfonate monooxygenase enzyme involves the carbon–sulfur bond cleavage of a wide range of organosulfonated compounds. A C4a-(hydro)peroxyflavin is the oxygenating intermediate in the mechanism of desulfonation by the alkanesulfonate monooxygenase. This review discusses the physiological importance of this system, and the individual kinetic parameters and mechanistic properties of this enzyme system.

Role of SsuF in the alkanesulfonate monooxygenase system from Pseudomonas putida

The alkanesulfonate monooxygenase system from Pseudomonas putida enables the organism to utilize sulfonates and sulfate esters from the environment. The alkanesulfonate monooxygenase system is comprised of an FMN reductase (SsuE) and flavin‐dependent monooxygenase (SsuD). Although this system from Escherichia coli has been studied, the alkanesulfonate system from P. putida has not been evaluated. The system in P. putida possesses a third protein, SsuF, but the role of SsuF in the desulfonation mechanism has not been determined. SsuF has shown to have a high identity to a molybdopterin binding protein within E. coli. Previous studies have shown that SsuF is required for growth with sulfate esters and sulfonates, but the exact function of this protein has not been determined.As P. putida possesses the additional SsuF protein, it is possible that the alkanesulfonate monooxygenase system may operate under an alternative mechanism than the E. coli system. Following the expression and purification of SsuF, several spectroscopic techniques were employed to analyze the structural and functional characteristics of the proteins. Further evaluation of SsuF was performed to determine the substrate range of SsuD and SsuE in the presence and absence of SsuF. Based on these studies, a functional role for SsuF in the desulfonation mechanism has been established.Work supported by NSF Grant MCB545048.

Investigating the Details of the Catalytic Mechanism of Alkanesulfonate Monooxygenase

Elemental sulfur is essential for the synthesis and functionality of certain biological cofactors and proteins. When excess sulfate is not readily available, Escherichia coli expresses a series of proteins implicated in the uptake of sulfur from alternative environmental sources. Notably among these sulfate‐starvation induced proteins is the two‐component alkanesulfonate monooxygenase system composed of an NAD(P)H‐dependent FMN reductase (SsuE) and a FMNH2‐dependent monooxygenase (SsuD). The details of the catalytic mechanism governing the desulfonation reaction of SsuD are under active investigation.Previous structural and kinetic studies have implicated an active site base serving a role in catalysis. Several possibilities for this active‐site base were identified, and mutagenesis was performed in order to assess the importance of each residue in catalysis. Kinetic Studies were performed to determine how these substitutions altered the kinetic parameters compared to wild type SsuD, and deuterium‐isotope studies were performed to evaluate further the mechanism of desulfonation. These results provided valuable insight into the overall catalytic mechanism of SsuD.

Catalytic Importance of the Substrate Binding Order for the FMNH2-Dependent Alkanesulfonate Monooxygenase Enzyme

The two-component alkanesulfonate monooxygenase system from Escherichia coli includes an FMN reductase (SsuE) and an FMNH2-dependent alkanesulfonate monooxygenase (SsuD) involved in the acquisition of sulfur from alkanesulfonates during sulfur starvation. The SsuD enzyme directly catalyzes the oxidation of alkanesulfonate to aldehyde and sulfite in the presence of O2 and FMNH2. The goal of these studies was to investigate the kinetic mechanism of SsuD through rapid reaction kinetics and substrate binding studies. The SsuD enzyme shows a clear preference for FMNH2 (Kd, 0.32 +/- 0.15 microM) compared to FMN (Kd, 10.2 +/- 0.4 microM) with a 1:1 binding stoichiometry for each form of the flavin. The kinetic trace of premixed SsuD and FMNH2 mixed with oxygenated buffer was best fit to a double exponential with no observed formation of the C4a-(hydro)peroxyflavin. However, when FMNH2 was mixed with SsuD and oxygenated buffer an initial fast phase (kobs, 12.9 s-1) was observed, suggesting that the mixing order is critical for the accumulation of the C4a-(hydro)peroxyflavin. Results from fluorimetric titrations with octanesulfonate imply that reduced flavin must bind first to promote octanesulfonate binding. When octanesulfonate was included in the kinetic studies the C4a-(hydro)peroxyflavin was observed at 370 nm when FMNH2 was not premixed with SsuD, which correlated with an increase in octanal product. There was a clear hyperbolic dependence on octanesulfonate binding, indicating that octanesulfonate binds in rapid equilibrium, and further results indicated there was a second isomerization step following binding. These results suggest that an ordered substrate binding mechanism is important in the desulfonation reaction by SsuD with reduced flavin binding first followed by either O2 or octanesulfonate.

Mechanism of flavin reduction in the alkanesulfonate monooxygenase system

The alkanesulfonate monooxygenase system from Escherichia coli is involved in scavenging sulfur from alkanesulfonates under sulfur starvation. An FMN reductase (SsuE) catalyzes the reduction of FMN by NADPH, and the reduced flavin is transferred to the monooxygenase (SsuD). Rapid reaction kinetic analyses were performed to define the microscopic steps involved in SsuE catalyzed flavin reduction. Results from single-wavelength analyses at 450 and 550 nm showed that reduction of FMN occurs in three distinct phases. Following a possible rapid equilibrium binding of FMN and NADPH to SsuE (MC-1) that occurs before the first detectable step, an initial fast phase (241 s − 1 ) corresponds to the interaction of NADPH with FMN (CT-1). The second phase is a slow conversion (11 s − 1 ) to form a charge–transfer complex of reduced FMNH 2 with NADP + (CT-2), and represents electron transfer from the pyridine nucleotide to the flavin. The third step (19 s − 1 ) is the decay of the charge–transfer complex to SsuE with bound products (MC-2) or product release from the CT-2 complex. Results from isotope studies with [(4 R)- 2H]NADPH demonstrates a rate-limiting step in electron transfer from NADPH to FMN, and may imply a partial rate-limiting step from CT-2 to MC-2 or the direct release of products from CT-2. While the utilization of flavin as a substrate by the alkanesulfonate monooxygenase system is novel, the mechanism for flavin reduction follows an analogous reaction path as standard flavoproteins.

Detection of Protein-Protein Interactions in the Alkanesulfonate Monooxygenase System from Escherichia coli

The two-component alkanesulfonate monooxygenase system utilizes reduced flavin as a substrate to catalyze a unique desulfonation reaction during times of sulfur starvation. The importance of protein-protein interactions in the mechanism of flavin transfer was analyzed in these studies. The results from affinity chromatography and cross-linking experiments support the formation of a stable complex between the flavin mononucleotide (FMN) reductase (SsuE) and monooxygenase (SsuD). Interactions between the two proteins do not lead to overall conformational changes in protein structure, as indicated by the results from circular dichroism spectroscopy in the far-UV region. However, subtle changes in the flavin environment of FMN-bound SsuE that occur in the presence of SsuD were identified by circular dichroism spectroscopy in the visible region. These data are supported by the results from fluorescent spectroscopy experiments, where a dissociation constant of 0.0022 +/- 0.0010 muM was obtained for the binding of SsuE to SsuD. Based on these studies, the stoichiometry for protein-protein interactions is proposed to involve a 1:1 monomeric association of SsuE with SsuD.

Identification of the Flavin Binding Site in the Flavin Reductase from the Alkanesulfonate Monooxygenase System in Escherichia coli

Sulfate starvation in Escherichia coli increases the synthesis of specific proteins which utilize alternate sulfur sources for growth. A tow-component enzyme system has been show to be involved in the conversion of alkanesulfonates to sulfides and the corresponding aldehyde. The system consists of two proteins, a flavin reductase (SsuE) and a monooxygenase (SsuD) which together catalyze the oxidation of alkanesulfonate to the products. Previous studies have identified a conserved amino acid sequence, RXFS, from an alignment search of E. coli flavin reductases. The Ser residue has been shown to be involved in flavin binding by interacting with the isoalloxazine ring at N(5). An alignment search of SsuE also identified a similar conserved amino acid sequence motif, R51FDS54, which may be involved in flavin binding. Ser54 from wild type SsuE was substituted with alanine and the SsuES54A protein purified. Results from circular dichroism spectroscopy of SsuES54A were essentially identical to wild type SsuE suggesing there were no major secondary structural changes due to this mutation. The km and kcat values for SsuES54A were 0.09 μM and 101.83 min−1 respectively. Compared with previous results from wild type SsuE, the km increased by 6-fold and the catalytic efficiency kcat/km decreased by 6.4-fold. In addition, the different kd value for SsuES54A compared to the wild type protein suggested this mutation altered the affinity of flavin binding. Our results show that Ser54 plays an essential role in flavin binding to SsuE. To further probe the flavin binding sites, studies involving multiple mutations of the conserved R51FDS54 region will be performed. This research is supported by Auburn University.

Reaction mechanism studies of alkanesulfonate monooxygenase from Escherichia coli

The two-component alkanesulfonate monooxygenase system from Escherichia coli belongs to a family of enzymes that utilizes FMN as a substrate rather than a bound prosthetic group. This two-component system that includes an FMN reductase (SsuE) and an FMNH2-dependent alkanesulfonate moonoxygenase (SsuD) are involved in the conversion of alkanesulfonate to sulfite and the corresponding aldehyde. The SsuD enzyme directly catalyzes the oxidation of alkanesulfonate to aldehyde and sulfite in the presence of FMNH2 and O2. The primary objective of this study is to elucidate the kinetic mechanism of this enzymatic reaction through rapid reaction kinetics. In the absence of alkanesulfonates, a 4a-(hydro)peroxyflavin intermediate has been identified by the reaction of premixed SsuD and reduced flavin with molecular oxygen. The formation of 4a-(hydro)peroxyflavin intermediate is oxygen-dependent with a second order rate constant of 2.35×104 M−1s−1. In the absence of alkanesulfonates, this complex decays to FMN and H2O2 at a second order rate of 1.67×104 M−1s−1. The pre-steady state results provide evidence for the involvement of a 4a-(hydro)peroxyflavin intermediate in the SsuD catalytic reaction. Current studies are focused on determining the FMNH2 binding affinity of SsuD and SsuE, and pre-steady state analysis of SsuD in the presence of alkanesulfonates. This research is supported by Auburn University.

Altered mechanism of the alkanesulfonate FMN reductase with the monooxygenase enzyme

The two-component alkanesulfonate monooxygenase system from Escherichia coli is comprised of an FMN reductase (SsuE) and a monooxygenase enzyme (SsuD) that together catalyze the oxidation of alkanesulfonate to the corresponding aldehyde and sulfite products. To determine the effects of protein interactions on catalysis, the steady-state kinetic parameters for SsuE were determined in single-enzyme assays and in the presence of the monooxygenase enzyme and alkanesulfonate substrate. In single-enzyme kinetic assays, SsuE followed an ordered sequential mechanism, with NADPH as the first substrate to bind and NADP + as the last product to dissociate. However, in the presence of SsuD and octanesulfonate the kinetic mechanism of SsuE is altered to a rapid equilibrium ordered mechanism, and the K m value for FMN is increased 10-fold. These results suggest that both the SsuD enzyme and alkanesulfonate substrate are required to ensure that the FMN reductase reaction proceeds to form the ternary complex with the subsequent generation of reduced flavin transfer.