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Functional Evaluation of the Π-Helix in the NAD(P)

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Functional Evaluation of the Π-Helix in the NAD(P) Article Cite This: Biochemistry 2018, 57, 4469−4477 pubs.acs.org/biochemistry Functional Evaluation of the π‑Helix in the NAD(P)H:FMN Reductase of the Alkanesulfonate Monooxygenase System Jonathan M. Musila,† Dianna L. Forbes, and Holly R. Ellis* Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849, United States *S Supporting Information ABSTRACT: A subgroup of enzymes in the NAD(P)H:FMN reduc- tase 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. acterial two-component FMN-dependent systems are com- family are comprised primarily of FMN-bound flavin reduc- B posed of a flavin reductase and a monooxygenase enzyme tases, but a subgroup of FMN reductases within the family are that catalyze the oxidation of diverse sulfonated substrates. The two-component monooxygenase systems that catalyze flavin trans- role of the flavin reductase is to supply reduced flavin to the fer. These two-component FMN reductases have a π-helical monooxygenase enzyme, while the monooxygenase enzyme uti- structure located at the tetramer interface (Figure 1A).6 This SsuE from https://pubs.acs.org/doi/10.1021/acs.biochem.8b00544. lizes the reduced flavin to activate molecular oxygen. The π-helix is generated by the insertion of a tyrosine residue Downloaded by AUBURN UNIV at 06:50:12:528 on May 27, 2019 two-component alkanesulfonate monooxygenase system is (Tyr118) in the conserved α4 helix that generates a change in upregulated in bacteria during sulfur starvation to cleave the intrastrand hydrogen bonding resulting in wide turns.6 Identi- − fi C S bonds of various alkanesulfonates generating sul te and fication of π-helices as discrete secondary structures is often 1 fi the corresponding aldehyde. The sul te generated is incor- overlooked when characterizing three-dimensional structures − porated into various sulfur-containing biomolecules. Interest- of proteins.7 10 Extensive structural analyses have determined ingly, some bacteria express additional two-component systems that π-helices in protein structures are often misannotated and under sulfur limitation to provide these organisms with a more − provide a distinct function in the majority of enzymes that 2 5 − complex mechanism for sulfur acquisition. The utilization contain this structure.7 10 The π-helical structures can modify of two-component systems for sulfur acquisition suggests a the conformation of an enzyme to provide alternative non- similar mechanism is utilized to ensure that cellular sulfur con- covalent contacts and/or destabilize the region to provide a centrations are maintained. Conserved structural features may gain of function. The conserved π-helices in structurally related optimize the catalytic properties of two-component FMN- flavin reductases of two-component systems suggest this sec- dependent systems. The structural and conformational changes of two-component monooxygenases must be synchronized to ondary structural element provides these enzymes with a com- fl fl mon function. simultaneously support avin reduction, reduced avin trans- fl fer, and the oxidation of substrates. However, the structural In the three-dimensional structure of SsuE with bound avin, properties that enable these flavin reductases to transfer the hydrogen bonding occurs between the hydrogen of the hydroxyl labile reduced flavin product to their monooxygenase partner group of Tyr118 and the oxygen atom backbone carbonyl of have not been fully evaluated. The SsuE FMN reductase of the alkanesulfonate mono- Received: May 14, 2018 oxygenase system belongs to the NAD(P)H:FMN reductase Revised: July 2, 2018 family based on a conserved flavodoxin fold.6 Enzymes in this Published: July 6, 2018 © 2018 American Chemical Society 4469 DOI: 10.1021/acs.biochem.8b00544 Biochemistry 2018, 57, 4469−4477 Biochemistry Article Figure 1. Structural analysis of the π-helix in SsuE. (A) Location of the π-helix (green) at the tetramer interface of SsuE. The black lines at the interface represent the contacts made between the π-helix of each monomer and the opposite monomer in the dimer pair. (B) Hydrogen bonding interactions between Tyr118 and the oxygen of the carbonyl group of Ala78 across the tetramer interface of SsuE. The amide group of Ala78 forms hydrogen bonding interactions with the C4 carbonyl oxygen of the bound flavin. (C) Observed π-stacking interactions between Tyr118 residues at the tetramer interface of SsuE. The Tyr118 π-stacking interactions occur at a diagonal between monomers of the dimer pair. The structures were rendered with Pymol using Protein Data Bank entry 4PTZ (SsuE).6 Ala78 across the tetramer interface (Figure 1B).6,11 The amide phosphate (monobasic anhydrous and dibasic anhydrous), nitrogen of Ala78 forms a hydrogen bond to the O4 atom of dimethyl sulfoxide (DMSO), 5,5-dithiobis(2-nitrobenzoic acid) FMN bound to the active site. Although not noted in the origi- (DTNB), lysozyme, and urea were from Sigma (St. Louis, MO). nal three-dimensional structure, Tyr118 also forms π-stacking Isopropyl β-D-1-thiogalactopyranoside (IPTG), sodium chloride, interactions with the Tyr118 residues across the tetramer and glycerol were obtained from Macron Fine Chemicals interface (Figure 1C). The SsuE tetramer interface region (Center Valley, PA). 1-Octanesulfonate was purchased from includes a region of the π-helix and comprises the protein− Fluka (Milwaukee, WI). protein interaction site with SsuD that facilitates flavin transfer. Site-Directed Mutagenesis and Purification of the Therefore, the tetramer of SsuE would need to undergo an Y118 SsuE Variants. Variants of Tyr118 in SsuE were gener- oligomeric shift to a dimer for this region of SsuE to effectively ated to investigate the roles of the π-helix in flavin reduction form protein−protein interactions with SsuD. In preliminary and transfer to SsuD and in the regulation of oligomeric-state investigations, substitution of Tyr118 with Ala resulted in a changes. The SsuE primers were designed as 27-base oligonu- 11 FMN-bound SsuE variant. Although the Y118A SsuE variant cleotides for the Y118S, Y118F, and ΔY118 SsuE variants. The fl fi fl retained avin speci city for FMN, the avin remained bound primers were ordered from Life Technologies by substituting even when reduced. The reduced Y118A SsuE variant showed TAT for Tyr with TCT or TTT or deleting the TAT codon for slow reactivity in NADPH oxidase assays, but supported elec- the Y118S, Y118F, or ΔY118 SsuE variant, respectively. The tron transfer to ferricyanide. In addition, there was no measur- Qiagen kit plasmid purification protocol was utilized to prepare fi able sul te production by SsuD when it was coupled with the SsuE plasmid used for site-directed mutagenesis. Following Y118A SsuE, which correlated with the inability of Y118A fi fl 11 site-directed mutagenesis, the SsuE variants were con rmed SsuE to support multiple avin reduction turnovers. Prelimi- through DNA sequencing analysis (Eurofins/Genomics, nary investigations of Y118A SsuE were important in providing Louisville, KY). The previously generated Y118A SsuE variant a basic foundation regarding the general properties of the was also utilized in these studies.11 The SsuD and SsuE enzymes π-helix. Additional Tyr118 SsuE variants were generated to fi π were expressed and puri ed from a pET21a expression vector in further evaluate the role of the -helix in promoting the oligo- E. coli strain BL21(DE3) as previously described.12 The protein meric changes in SsuE that promote flavin transfer. fractions for the flavin-free SsuE enzymes and SsuD were collected based on the ultraviolet−visible (UV−vis) absorb- ■ EXPERIMENTAL PROCEDURES ance at 280 nm. For the flavin-containing SsuE variants, the Materials. Escherichia coli strains [XL-1 Blue and BL21(DE3)] fractions were collected based on the UV−vis absorbance at were purchased from Stratagene (La Jolla, CA). Plasmid vectors 457 nm. Circular dichroism spectroscopy to evaluate the overall and pET21a were obtained from Novagen (Madison, WI). DNA gross secondary structure of the variants was performed as primers were synthesized by Invitrogen (Carlsbad, CA). Ampi- previously outlined.13 cillin, streptomycin sulfate, lysozyme, potassium bromide, ammo- Kinetic Analysis
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