Dodecin is the key player in flavin homeostasis of archaea

Abstract

Flavins are employed to transform physical input into biological output signals. In this function, flavins catalyze a variety of light-induced reactions and redox processes. However, nature also provides flavoproteins with the ability to uncouple the mediation of signals. Such proteins are the riboflavin-binding proteins (RfBPs) with their function to store riboflavin for fast delivery of FMN and FAD. Here we present in vitro and in vivo data showing that the recently discovered archaeal dodecin is an RfBP, and we reveal that riboflavin storage is not restricted to eukaryotes. However, the function of the prokaryotic RfBP dodecin seems to be adapted to the requirement of a monocellular organism. While in eukaryotes RfBPs are involved in trafficking riboflavin, and dodecin is responsible for the flavin homeostasis of the cell. Although only 68 amino acids in length, dodecin is of high functional versatility in neutralizing riboflavin to protect the cellular environment from uncontrolled flavin reactivity. Besides the predominant ultrafast quenching of excited states, dodecin prevents light-induced riboflavin reactivity by the selective degradation of riboflavin to lumichrome. Coordinated with the high affinity for lumichrome, the directed degradation reaction is neutral to the cellular environment and provides an alternative pathway for suppressing uncontrolled riboflavin reactivity. Intriguingly, the different structural and functional properties of a homologous bacterial dodecin suggest that dodecin has different roles in different kingdoms of life.

FIGURE 1.

Flavin biosynthesis. GTP and ribulose 5-phosphate are the educts in flavin synthesis. The key step in flavin biosynthesis is the dismutase reaction catalyzed by riboflavin synthase (a), building the tricyclic heteronuclear organic ring isoalloxazine. RF is further converted into FMN by riboflavin kinase (b) and subsequently transformed into FAD by FAD synthase (c). The catalytically active isoalloxazine is highlighted by a gray rectangle in the RF chemical structure. For a more detailed description of the flavin biosynthesis see supplemental Fig. 1.

FIGURE 2.

Comparison of HsDod^A and HhDod^B. A, superposition of the C-α traces of HsDod^A (red) and HhDod^B (blue) as separate monomers and as monomers part of the dodecameric assembly (superposition on the HsDod^A C-α trace in gray with bound riboflavins in color code of monomers). View 1 shows dodecin oriented along a 3-fold axis and view 2 along a 2-fold axis. HsDod^A labels are in black and HhDod^B in gray. B, structure-based sequence alignment and C-α root mean square deviation between the archaeal HsDod^A and the bacterial HhDod^B. The largest structural deviations in C-α atoms between HsDod^A and HhDod^B involve residues 47–52 (50–55 of HhDod^B). This peak corresponds to differences in the loop between theβ2 andβ3 strands. C, inverted binding of RF in HsDod^A (red) and in HhDod^B (blue). Trp³⁶ (W36) and Gln⁵⁵ (Q55), clamping and aligning the isoalloxazine rings in tetrade arrangements, are shown (HsDod^A numbering). In contrast to HsDod^A, the glutamine in HhDod^B binds the flipped flavin of the dimer-related monomer. In both structures, the distance between the indole and the isoalloxazine aromatic rings is about 3.3 Å. D, deviation in the main chain architecture to meet the different requirements of ligand incorporation. The widened pocket of HsDod^A allows accommodation of the dimethylated benzene ring in the re-re arranged aromatic tetrade (see minor peak in Fig. 2B between residues 42 and 46). Arg⁴⁶ (R46) of HhDod^B, highly conserved in bacterial dodecins, accounts for hydrogen bonding to the isoalloxazine moiety. This isoalloxazine-protein interaction is missing in HsDod^A. For clarity, side chains of HsDod^A are omitted, and the binding pocket is reduced to the 2-fold related part.

FIGURE 3.

Light-induced degradation of free and dodecin-bound RF. A, illumination of free and dodecin-bound RF. Chromatographic profiles of RF/HsDod^A (HsDod^A_i-HsDod^A_iii) and RF/HhDod^B (HhDod^B_i-HhDod^B_iii) at 0 min and after 4 h of illumination with light. Fluorescence detection is performed at 520 (i, ii) and 464 nm (iii). Chemical structures for RF (○) and LCR (□) are shown as insets in fluorescence lanes. Note that the EU scale is different in lanes i–iii. B, molar concentrations of RF fitted to a first order decay; HsDod^A (black circle, solid line) and HhDod^B (gray circle, gray solid line) and free RF shown in inset. Half-lives were determined to be 32 ± 1.4 s (free RF), 1180 ± 180 s (19.7 min; HsDod^A) and 1480 ± 230 s (24.7 min, HhDod^B), respectively. C, light stability of dodecin-bound RF (in air). Concentrations of RF and LCR incorporated into HsDod^A (•/▪) and HhDod^B (○/□) and the respective overall concentrations (▴/Δ) are shown.

FIGURE 4.

Time-resolved spectroscopic characterization of free and dodecin-bound RF. A, excitation of RF (panel i), RF incorporated in HsDod^A (RF/HsDod^A) (panel ii), and in HhDod^B (RF/HhDod^B) (panel iii) performed at 388 nm with sapphire white light as the probing pulse. Positive absorbance changes are shown in red and negative in blue. B, transient absorbance changes at 451 nm. Free RF is shown as a dashed line in black, the corresponding RF/HsDod^A and RF/HhDod^B complexes in black and gray solid lines, respectively.

FIGURE 5.

Flavin and LCR concentrations during H. salinarum growth. A, growth curves of H. salinarum strains R1 (wild type) and R1Δdod (dodecin deleted). The color code of growth phases (exponential, yellow; early stationary, orange; late stationary, red) is used in B and C, and Fig. 7A. B, flavin concentrations during H. salinarum growth. Quantitative analysis of intracellular flavin reveals similar FMN (star) and FAD (Δ) concentrations in wild type cells cultured in the dark (R1_d, black) and in the light (R1_l, red), as well as in the deletion strain R1Δdod in the dark (R1Δdod_d, gray). Concentrations of RF (○) show major differences with respect to light and dodecin. C, LCR concentrations during H. salinarum growth. LCR molar concentrations are about 10-fold increased in the H. salinarum wild type strain R1 grown in light (R1_l, red) as compared with cells grown in the dark (R1_d, black) or depleted in dodecin (R1Δdod_d, gray). LCR concentrations were below the detection limit (<0.5 μm) in the R1Δdod_d inoculation culture and at day 4 in cultures R1_d and R1Δdod_d.

FIGURE 6.

Dodecin protein and mRNA levels during growth of H. salinarum. A, Western blot analysis of dodecin in R1 wild type strain grown in the dark (R1_d) and in light (R1_l). B, analysis of the dodecin mRNA levels in cells of R1_d (▪), R1_l (•, dashed line), and R1Δdod_d (gray triangle) in the time period of the rather constant dodecin expression observed in A. A 2^{-ΔΔCt(xi-x4)} value of 1 means no difference in the dodecin mRNA level at the respective day compared with reference day 4; ½ and 2, a 2-fold down-regulation and up-regulation, respectively. 2^-ΔΔCt(l-d) values illustrate the differences in dodecin mRNA levels when cells grown in light (R1_l) and in dark (R1_d).

FIGURE 7.

Working scheme of the archaeal dodecin on the cellular and on the protein level. A, dodecin cycle. Dodecin expression is induced in the exponential phase. Although present, the uptake of FMN and FAD is prevented by their low affinity (stage 1). In the stationary phase, RF (orange) from degradation of flavins and/or biosynthesis is sequestered by dodecin, establishing a flavin buffer (stage 2). Stored RF is available for biosynthesis of FMN and FAD when favorable condition induced metabolic activity (early exponential growth phase, stage 3). Intracellular flavin concentrations are given in insets. Attached schemes abstract the interplay of free RF, bound RF (RF/HsDod^A), and converted RF (FMN/FAD). B, working modes of HsDod^A. Shown is a model for the nondestructive quenching (working mode 1, green) and the destructive dealkylation reaction (working mode 2, red) as parallel reactions. Photoreduction and photodealkylation are reported to lead to LCR as a degradation product. Because of the high affinity of HsDod^A for LCR, LCR is kept captured in the binding pocket. The intermediate state in working mode 1 is shown as a charged radical pair prior to a putative protonation/deprotonation step.

FIGURE 8.

Rampp plot of dodecin homologous sequences. 107 sequences identified as homologs of HsDod^A are listed according to standard taxonomy of the respective species and blasted against each other. E-values are arranged in a two-dimensional matrix with colors representing the conservation according to the legend at left. Each pixel represents the E-value of the sequence pair at the respective horizontal and vertical axis. The archaeal subfamily clusters as a highly homologous group of sequences, with significantly lower similarity to bacterial dodecins. This is indicated by a dark frame with a bright spot along the diagonal (for a list of organism see supplemental Fig. 6 and Fig. 7).