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

Abstract

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.

Oligomeric Changes Regulate Flavin Transfer in Two-Component FMN Reductases Involved in Sulfur Metabolism

“…The fluorescence decrease observed at higher pyridine nucleotide concentrations is due to unbound NADPH in solution . MsuE and the MsuD monooxygenase partner had a distinct specificity for FMN and NADPH in coupled assays with the MsuD monooxygenase partner, but the specificity of MsuE for each substrate in the absence of MsuD had not been determined . MsuE showed a similar affinity for FMN/FAD and NADH/NADPH, indicating there was no apparent preference for a specific substrate under equilibrium conditions (Figure C,D, Table , and Figure S1C,D).…”

“…The principal structural difference between the two groups of enzymes is the presence of a π-helix located at the dimer/dimer interface. 10,21,37,38 It has been proposed that the π-helix structure enables SsuE to transfer flavin through protein−protein interactions to the SsuD monooxygenase partner. [11][12][13]39 The SsuE−SsuD interaction sites are located at the dimer/dimer interface of SsuE so the enzyme would need to undergo a conformational change to form these interactions.…”

“…All reagents were purchased from Sigma-Aldrich, Biorad, or Fisher. The SsuE and MsuE genes were cloned separately into the RNA polymerase-dependent vector pET21a (Novagen) and expressed in Escherichia coli BL21 (DE3), as previously described. , The concentrations of SsuE and MsuE in solution were calculated with the molar extinction coefficient of each protein (20.3 and 7.4 mM –1 cm –1 ). , …”

Shorter Alkanesulfonate Carbon Chains Destabilize the Active Site Architecture of SsuD for Desulfonation

“…The concentrations of MsuD and MsuE proteins were determined from A 280 measurements using molar extinction coefficients of 49.4 and 7.54 mM −1 cm −1 , respectively. 17,18 Steady-State Kinetic Analyses. A coupled assay monitoring sulfite production was used to determine the steadystate kinetic parameters of SsuD and MsuD.…”

“…SsuD, SsuE, MsuD, and MsuE enzymes were purified as described previously. , The concentrations of SsuD and SsuE proteins were determined from A 280 measurements using molar extinction coefficients of 47.9 and 20.3 mM –1 cm –1 , respectively. The concentrations of MsuD and MsuE proteins were determined from A 280 measurements using molar extinction coefficients of 49.4 and 7.54 mM –1 cm –1 , respectively. , …”