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.

The alkanesulfonate monooxygenase enzymes (SsuE and SsuD) catalyze the desulfonation of diverse alkanesulfonate substrates. The SsuE enzyme is an NADPH-dependent FMN reductase that provides reduced flavin to the SsuD monooxygenase enzyme. Previous studies have highlighted the presence of protein–protein interactions between SsuE and SsuD thought to be important in the flavin transfer event, but the putative interaction sites have not been identified. Protected sites on specific regions of SsuE and SsuD were identified by hydrogen–deuterium exchange mass spectrometry. An α-helix on SsuD containing conserved charged amino acids showed a decrease in percent deuteration in the presence of SsuE. The α-helical region of SsuD is part of an insertion sequence and is adjacent to the active site opening. A SsuD variant containing substitutions of the charged residues showed a 4-fold decrease in coupled assays that included SsuE to provide reduced FMN, but there was no activity observed with an SsuD variant containing a deletion of the α-helix under similar conditions. Desulfonation by the SsuD deletion variant was only observed with an increase in enzyme and substrate concentrations. Although activity was observed under certain conditions, there were no protein–protein interactions observed with the SsuD variants and SsuE in pull-down assays and fluorimetric titrations. The results from these studies suggest that optimal transfer of reduced flavin from SsuE to SsuD requires defined protein–protein interactions, but diffusion can occur under specified conditions. A basis is established for further studies to evaluate the structural features of the alkanesulfonate monooxygenase enzymes that promote desulfonation.

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 D2Okcat of 0.75 ± 0.04 with no observable effect on D2Okcat/Km. This resulted in equivalent solvent isotope effects on D2Okcat 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.

The alkanesulfonate monooxygenase enzyme (SsuD) catalyzes the oxygenolytic cleavage of a carbon–sulfur bond from sulfonated substrates. A mechanism involving acid–base catalysis has been proposed for the desulfonation mechanism by SsuD. In the proposed mechanism, base catalysis is involved in abstracting a proton from the alkane peroxyflavin intermediate, while acid catalysis is needed for the protonation of the FMNO– intermediate. The pH profiles of kcat indicate that catalysis by SsuD requires a group with a pKa of 6.6 ± 0.2 to be deprotonated and a second group with a pKa of 9.5 ± 0.1 to be protonated. The upper pKa value was not present in the pH profiles of kcat/Km. Several conserved amino acid residues (His228, His11, His333, Cys54, and Arg226) have been identified as having potential catalytic importance due to the similar spatial arrangements with close structural and functional relatives of SsuD. Substitutions to these amino acid residues were generated, and the pH dependencies were evaluated and compared to wild-type SsuD. Although a histidine residue was previously proposed to be the active site base, the His variants possessed similar steady-state kinetic parameters as wild-type SsuD. Interestingly, R226A and R226K SsuD variants possessed undetectable activity, and there was no detectable formation of the C4a-(hydro)peroxyflavin intermediate for the Arg226 SsuD variants. Guanidinium rescue with the R226A SsuD variant resulted in the recovery of 1.5% of the wild-type SsuD kcat value. These results implicate Arg226 playing a critical role in catalysis and provide essential insights into the mechanistic steps that guide the SsuD desulfonation process.

The structure of the flavin-dependent alkanesulfonate monooxygenase (SsuD) exists as a TIM-barrel structure with an insertion region located over the active site that contains a conserved arginine (Arg297) residue present in all SsuD homologues. Substitution of Arg297 with alanine (R297A SsuD) or lysine (R297K SsuD) was performed to determine the functional role of this conserved residue in SsuD catalysis. While the more conservative R297K SsuD possessed a lower kcat/Km value (0.04 ± 0.01 μM–1 min–1) relative to wild-type (1.17 ± 0.22 μM–1 min–1), there was no activity observed with the R297A SsuD variant. Each of the arginine variants had similar Kd values for flavin binding as wild-type SsuD (0.32 ± 0.15 μM), but there was no measurable binding of octanesulfonate. The low levels of activity for the R297A and R297K SsuD variants correlated with the absence of any detectable C4a-(peroxy)flavin formation in stopped-flow kinetic studies. Single-turnover experiments were performed in the presence of SsuE to evaluate both the reductive and oxidative half-reaction. With wild-type SsuD a lag phase is observed following the reductive half-reaction by SsuE that represents flavin transfer or conformational changes associated with the binding of substrates. Evaluation of the Arg297 SsuD variants in the presence of SsuE showed no lag phase following reduction by SsuE, and the flavin was oxidized immediately following the reductive half-reaction. These results corresponded with a lack of detectable changes in the proteolytic susceptibility of R297A and R297K SsuD in the presence of reduced flavin and/or octanesulfonate, signifying the absence of a conformational change in these variants with the substitution of Arg297.

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.Graphical abstractHighlights► In this review we examine the mechanism of the alkanesulfonate monooxygenase system. ► Flavin reduction by SsuE follows an order sequential mechanism. ► The SsuE mechanism is altered in the presence of SsuD and octanesulfonate. ► A C4a-(hydro)peroxyflavin is the oxygenating flavin intermediate in the SsuD reaction. ► Two mechanistic strategies for desulfonation by SsuD are described.

Detailed kinetic studies were performed in order to determine the role of the single cysteine residue in the desulfonation reaction catalyzed by SsuD. Mutation of the conserved cysteine at position 54 in SsuD to either serine or alanine had little effect on FMNH2 binding. The kcat/Km value for the C54S SsuD variant increased 3-fold, whereas the kcat/Km value for C54A SsuD decreased 6-fold relative to wild-type SsuD. An initial fast phase was observed in kinetic traces obtained for the oxidation of flavin at 370 nm when FMNH2 was mixed against C54S SsuD (kobs, 111 s− 1) in oxygenated buffer that was 10-fold faster than wild-type SsuD (kobs, 12.9 s− 1). However, there was no initial fast phase observed in similar kinetic traces obtained for C54A SsuD. This initial fast phase was previously assigned to the formation of the C4a-(hydro)peroxyflavin in studies with wild-type SsuD. There was no evidence for the formation of the C4a-(hydro)peroxyflavin with either SsuD variant when octanesulfonate was included in rapid reaction kinetic studies, even at low octanesulfonate concentrations. The absence of any C4a-(hydro)peroxyflavin accumulation correlates with the increased catalytic activity of C54S SsuD. These results suggest that the conservative serine substitution is able to effectively take the place of cysteine in catalysis. Conversely, decreased accumulation of the C4a-(hydro)peroxyflavin intermediate with the C54A SsuD variant may be due to decreased activity. The data described suggest that Cys54 in SsuD may be either directly or indirectly involved in stabilizing the C4a-(hydro)peroxyflavin intermediate formed during catalysis through hydrogen bonding interactions.

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 FMNH2 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 [(4R)-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.