Structural Features That Promote Catalysis in Two-Component Systems Involved in Sulfur Metabolism

Abstract

Sulfur is an essential element important in the synthesis of biomolecules. Bacteria are able to assimilate inorganic sulfur for the biosynthesis of L-cysteine. Inorganic sulfate is often unavailable, so bacteria have evolved multiple metabolic pathways to obtain sulfur from alternative sources. Interestingly, many of the enzymes involved in sulfur acquisition are flavin-dependent two-component systems. These two-component systems consist of a flavin reductase and monooxygenase that utilize flavin to cleave the carbon-sulfur bonds of organosulfur compounds. The two-component systems differ in their characterized sulfur substrate specificity. Enzymes SsuE/SsuD are involved in the desulfonation of linear alkanesulfonates (C2-C10), enzymes MsuE/MsuD utilize methanesulfonate (C1), and enzymes SfnF/SfnG utilize DMSO2 as a sulfur source. The flavin reductases involved in sulfur assimilation utilize FMN as a substrate but differ in their ability to utilize NADH or NADPH. The alkanesulfonate monooxygenase system was the first two-component flavin-dependent system expressed during sulfur limiting conditions that was characterized. The flavin reductase (SsuE) and monooxygenase (SsuD) have distinct structural and functional properties, but the two enzymes must synchronize their functions for catalysis to occur. Once flavin is reduced, protein-protein interactions between the SsuE and SsuD facilitate reduced flavin to protect reactive intermediates from bulk solvent. The flavin reductase undergoes an oligomeric conversion from a tetramer to a dimer in the presence of flavin or SsuD. These oligomeric changes have been proposed to promote protein-protein interactions and flavin transfer. The π-helix is a conserved structural feature of all the flavin reductases in these two-component systems, that was initially proposed to be generated by a single amino insertion into a conserved α-helical region. The π-helix in proteins often provide a gain of function for the enzyme. The π- iii helix in SsuE is formed by the insertion of a Tyr residue (Tyr118) into a conserved α-helix. The π-helix has been proposed to play a role in the observed oligomeric conversion from a tetramer to a dimer needed for protein-protein interactions between SsuE and SsuD. The ability of other FMN reductases (MsuE and SfnE) to undergo this conversion is currently unknown. Interestingly, both MsuE and SfnE contain a similar π-helix with a histidine insertional residue in the π-helix. A goal of these research studies was to identify common functional features among the π-helices in two-component FMN reductases and determine how they differ from canonical flavin reductases. Variants of Tyr118 were generated and their three-dimensional 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 Try118 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 crystallized as a dimer. Single amino acid substitutions of Y118 to His in SsuE and MsuE (H126) were generated to determine if the variants would maintain the functional attributes of the wild-type enzymes. Exchanging the π-helix insertional residue of each enzyme did not result in the expected, equivalent kinetic properties. The His126 conversion to Tyr in MsuE did not change the kinetic properties of the enzyme and the variant was able to provide reduced flavin to both MsuD and SsuD. Conversely, the Y118H SsuE variant did not possess reductase activity, and was unable to support flavin transfer to MsuD or SsuD. Structure-based sequence analysis further demonstrated the presence of a similar Tyr residue in a FMN-bound reductase in the NADPH:FMN reductase family that is not sufficient to generate a π-helix. Results from structural and functional studies of iv 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. Further analysis identified a structurally similar enzyme, ChrR, in the same family that has a similar residue Tyr126 in similar location as Try118 of SsuE. ChrR has two glutamate residues located in similar position as conserved residues proline and aspartic acid for flavin reductases. These conserved residues may play a role in stabilizing the π-helical region for flavin reductases. Results from the variants generated suggest the residues play a role in stabilizing the overall oligomeric structure due to the low success during purification and unsuccessful transfer of substrates to partner enzymes. The monooxygenases SsuD, MsuD, and SfnG catalyze the desulfonation of organosulfur substrates. High amino acid sequence identity between SsuD and MsuD suggest they utilize similar structural and functional features for catalysis. The SsuD enzyme has a TIM-barrel fold, but diverges from classic TIM-barrel structures due to insertional regions. This SsuD insertional region contains a long loop region that protrudes over the active site. Once substrates are transferred to the monooxygenase, this mobile loop interacts with substrate to initiate conformational changes that protect reactive intermediates. Arg297 is located on the mobile loop and previous studies suggest the amino acid forms interactions the flavin phosphate. Similar interactions have been observed in TIM-barrel enzymes. These structural features may play a role in substrate specificity and protect reactive intermediates from solvent for FMN-dependent two-component systems. FMN substrate fragments were used to evaluate the role of the phosphate group in assisting in loop closure. There was no activity observed with riboflavin and increasing phosphite concentrations as the substrate, suggesting the loop stabilization may not occur with the FMN phosphate alone. v The monooxygenase enzymes involved in the desulfonation of alkanesulfonates have been proposed to have different substrate specifies. The MsuD enzyme has been proposed to have a substrate preference for methanesulfonate, while SsuD shows a preference for alkanesulfonates between C6-C10 carbons. The SsuD and MsuD enzymes share ~60% amino acid sequence identity. Structural models of MsuD suggested they likely share similar active site architectures. Therefore, it was unclear what structural features contribute to the substrate specificity. In coupled assays, MsuD was able to utilize a wide range of alkanesulfonate substrates including methanesulfonate. SsuD was able to utilize similar substrates as MsuD, but was unable to catalyze the desulfonation of methanesulfonate. The inability of SsuD to utilize methanesulfonate agrees with previous results, but is curious given their nearly identical active sites. The results from these described studies have provided important information on the structural features conserved in two-component monooxygenase enzymes that determine specific functions. It is clear that flavin transfer in these enzymes creates an added challenge for these two component flavin-dependent systems. While these enzymes share similar structures and functions, they have evolved to maintain their own distinctive features for catalytic function. These differences would provide bacteria with more diverse processes for sulfur acquisition.