Figure 1. Dihydrouridine biosynthesis and localization of D residues in the tRNA. (A) Reduction reaction of uridine to dihydrouridine in tRNAs and mRNAs catalyzed by dihydrouridine synthases (Dus). These proteins are flavoenzymes that use FMN as a redox coenzyme and NADPH as a reductant source. (B) Cloverleaf secondary structure of the tRNAs shows the location of the D residues as well as the Dus enzymes that introduce them into Escherichia coli, Mycoplasma capricolum, and Saccharomyces cerevisiae.

Figure 1. Dihydrouridine biosynthesis and localization of D residues in the tRNA. (A) Reduction reaction of uridine to dihydrouridine in tRNAs and mRNAs catalyzed by dihydrouridine synthases (Dus). These proteins are flavoenzymes that use FMN as a redox coenzyme and NADPH as a reductant source. (B) Cloverleaf secondary structure of the tRNAs shows the location of the D residues as well as the Dus enzymes that introduce them into Escherichia coli, Mycoplasma capricolum, and Saccharomyces cerevisiae.

Figure 2. Crystallographic structures of Dus. (A,B) crystal structures of T. thermophilus DusA (PDB: 3B0V), human Dus2 without the dsRBD (PDB: 4XP7), and the dsRBD of hDus2 (PDB: 4WFT), respectively. The TIM-barrel domain (TBD) appears in teal, while the helical domain (HD) is in blue, the inserted beta-sheet in red, the connecting alpha-helix (c-αH) in green, and the dsRBD in purple. The FMN coenzyme is denoted in yellow. Above each of the structures is a scheme of the modular organization of Dus2, in which the delineation of each domain is shown.

Figure 2. Crystallographic structures of Dus. (A,B) crystal structures of T. thermophilus DusA (PDB: 3B0V), human Dus2 without the dsRBD (PDB: 4XP7), and the dsRBD of hDus2 (PDB: 4WFT), respectively. The TIM-barrel domain (TBD) appears in teal, while the helical domain (HD) is in blue, the inserted beta-sheet in red, the connecting alpha-helix (c-αH) in green, and the dsRBD in purple. The FMN coenzyme is denoted in yellow. Above each of the structures is a scheme of the modular organization of Dus2, in which the delineation of each domain is shown.

Figure 3. Phylogenetic tree of 280 Dus2 family members illustrating domain architecture diversity. Moving from the inside of the figure to the outside: (1) Sequence lengths are denoted by the central histogram; (2) Colored bars (one square per organism) indicate the presence of distinct fusion proteins (note: given multiple squares for one organism, they indicate the instance of separately encoded homologs); (3) Percent coverage of sequences for both the remaining lengths not identified as belonging to any recognizable domain (inside) and the identified Dus domain (outside) are shown. A red asterisk among the labels of the tree’s leaves denotes the zinc finger domain-containing homolog of Pythium insidiosum that, in addition to ZnD_U1, also contains a separate ICL_KPHMT fusion domain.

Figure 3. Phylogenetic tree of 280 Dus2 family members illustrating domain architecture diversity. Moving from the inside of the figure to the outside: (1) Sequence lengths are denoted by the central histogram; (2) Colored bars (one square per organism) indicate the presence of distinct fusion proteins (note: given multiple squares for one organism, they indicate the instance of separately encoded homologs); (3) Percent coverage of sequences for both the remaining lengths not identified as belonging to any recognizable domain (inside) and the identified Dus domain (outside) are shown. A red asterisk among the labels of the tree’s leaves denotes the zinc finger domain-containing homolog of Pythium insidiosum that, in addition to ZnD_U1, also contains a separate ICL_KPHMT fusion domain.

Figure 4. 3D structural models of various Dus2. (A) Models of Dus2 showing minimal modularity. The TIM-barrel domain (TBD) appears in teal, while the inserted beta-sheet is in red, the helical domain (HD) is in blue, the connective alpha-helix (c-αH) is in green, and the other structural extensions are in pink. The FMN coenzyme is denoted in yellow. (B) Models of Dus2 showing complex modularity with the addition of an extra domain. The same color codes for the canonical domains are followed (TBD + inserted beta sheet + HD). Rossman, zinc finger, dsRBD, PyrOX_2, ICL_KPHMT, and CTNS domains are in yellow, pink, purple, light green, orange, and olive, respectively. Above each of model is represented the schematic modular organization of Dus2 and is indicated the boundary of each domain. We have chosen not to show the delineation of the inserted beta-sheet in order to avoid figure overload. However, this structural element is colored in red in each of the 3D models presented.

Figure 4. 3D structural models of various Dus2. (A) Models of Dus2 showing minimal modularity. The TIM-barrel domain (TBD) appears in teal, while the inserted beta-sheet is in red, the helical domain (HD) is in blue, the connective alpha-helix (c-αH) is in green, and the other structural extensions are in pink. The FMN coenzyme is denoted in yellow. (B) Models of Dus2 showing complex modularity with the addition of an extra domain. The same color codes for the canonical domains are followed (TBD + inserted beta sheet + HD). Rossman, zinc finger, dsRBD, PyrOX_2, ICL_KPHMT, and CTNS domains are in yellow, pink, purple, light green, orange, and olive, respectively. Above each of model is represented the schematic modular organization of Dus2 and is indicated the boundary of each domain. We have chosen not to show the delineation of the inserted beta-sheet in order to avoid figure overload. However, this structural element is colored in red in each of the 3D models presented.

Figure 5. Modularity and domain orientation in Dus2. (A) Structural 3D model of A. queenslandica Dus2. The canonical domains are in gray while the c-αH and the dsRBD are in red. The linker attaching the c-αH to the dsRBD is indicated. (B) Structural superposition of the 5 A. queenslandica Dus2 model generated by AlphaFold2. The TIM-barrel and the HD of each model are in gray while the dsRBD are colored in a different color for each model. (C) Structural 3D model of M. pennsylcanicum Dus2 showing to different view that differs by a rotation of 180° around the z-axis. The canonical domains are in gray while the c-αH and PyrOx domains are in red. (D) Structural superposition of models 1 and 2 of M. pennsylcanicum Dus2 generated by AlphaFold2. Model 1 is colored as indicated in (C) while model 2 is in cyan. The double-headed arrow indicates the different orientation of the PyrOx domain in the two models. (E) Structural 3D model of P. insidiosum Dus2. The canonical domains are in gray while the c-αH, the ZnFD, and the ICL-KPHMT domains are in red. (F) Structural superposition of models 1 and 2 of P. insidiosum Dus2 generated by AlphaFold2. Model 1 is colored as indicated in (E) while model 2 is in cyan. The double-headed arrows indicate the difference in orientation of the ZnFD and ICL-KPHMT domains in the two models. (G) Structural 3D model of T. nelsoni Dus2. The canonical domains are in gray while the c-αH, the dsRBD, and the CTNS domains are in red. (H) Structural superposition of models 1 and 2 of T. nelsoni Dus2 generated by AlphaFold2. Model 1 is colored as indicated in (G) while model 2 is in cyan. The double-headed arrows indicate the respective difference in orientation of the dsRBD and CTNS domains in the two models.

Figure 5. Modularity and domain orientation in Dus2. (A) Structural 3D model of A. queenslandica Dus2. The canonical domains are in gray while the c-αH and the dsRBD are in red. The linker attaching the c-αH to the dsRBD is indicated. (B) Structural superposition of the 5 A. queenslandica Dus2 model generated by AlphaFold2. The TIM-barrel and the HD of each model are in gray while the dsRBD are colored in a different color for each model. (C) Structural 3D model of M. pennsylcanicum Dus2 showing to different view that differs by a rotation of 180° around the z-axis. The canonical domains are in gray while the c-αH and PyrOx domains are in red. (D) Structural superposition of models 1 and 2 of M. pennsylcanicum Dus2 generated by AlphaFold2. Model 1 is colored as indicated in (C) while model 2 is in cyan. The double-headed arrow indicates the different orientation of the PyrOx domain in the two models. (E) Structural 3D model of P. insidiosum Dus2. The canonical domains are in gray while the c-αH, the ZnFD, and the ICL-KPHMT domains are in red. (F) Structural superposition of models 1 and 2 of P. insidiosum Dus2 generated by AlphaFold2. Model 1 is colored as indicated in (E) while model 2 is in cyan. The double-headed arrows indicate the difference in orientation of the ZnFD and ICL-KPHMT domains in the two models. (G) Structural 3D model of T. nelsoni Dus2. The canonical domains are in gray while the c-αH, the dsRBD, and the CTNS domains are in red. (H) Structural superposition of models 1 and 2 of T. nelsoni Dus2 generated by AlphaFold2. Model 1 is colored as indicated in (G) while model 2 is in cyan. The double-headed arrows indicate the respective difference in orientation of the dsRBD and CTNS domains in the two models.

Figure 6. The Zinc finger domain in Dus2. (A) Structural 3D model of A. candida Dus2. The canonical domains are in gray while the c-αH and the ZnFD are in green and red, respectively. (B) Structural 3D model of S. parasitica Dus2. (C,D) Zoom on the zinc finger motifs of A. candida and S. parasitica Dus2, respectively. The signature of the motif is indicated in the red box. (E) Solution NMR structure of a ZnFD of human JAZ protein (PDB: 2MKN). The zinc atom is represented as a ball colored in cyan. The ZnF motif for this domain is indicated below the structure. (F) Structural superposition of 2MKD (colored in purple) with the ZnFD of S. parasitica Dus2 (in red). The ZnF motif of S. parasitica Dus2 is shown below the figure of the structural alignment. (G) Structural superposition of 2MKN (PDB code for the structure of JAZ ZnFD in complex with a dsRNA with the ZnFD of S. parasitica Dus2 (in red). Two different views that differ by a rotation of 180° around the z-axis are shown. (H) Model of the S. parastica Dus2/dsRNA complex. The ZnFD is represented in the electrostatic surface mode.

Figure 6. The Zinc finger domain in Dus2. (A) Structural 3D model of A. candida Dus2. The canonical domains are in gray while the c-αH and the ZnFD are in green and red, respectively. (B) Structural 3D model of S. parasitica Dus2. (C,D) Zoom on the zinc finger motifs of A. candida and S. parasitica Dus2, respectively. The signature of the motif is indicated in the red box. (E) Solution NMR structure of a ZnFD of human JAZ protein (PDB: 2MKN). The zinc atom is represented as a ball colored in cyan. The ZnF motif for this domain is indicated below the structure. (F) Structural superposition of 2MKD (colored in purple) with the ZnFD of S. parasitica Dus2 (in red). The ZnF motif of S. parasitica Dus2 is shown below the figure of the structural alignment. (G) Structural superposition of 2MKN (PDB code for the structure of JAZ ZnFD in complex with a dsRNA with the ZnFD of S. parasitica Dus2 (in red). Two different views that differ by a rotation of 180° around the z-axis are shown. (H) Model of the S. parastica Dus2/dsRNA complex. The ZnFD is represented in the electrostatic surface mode.

Figure 7. Structural comparison of the ZnFD of Dus2 and that of S. cerevisiae Mod5. ZnFD of S. parasitica Dus2 (in red) is superimposed on the ZnFD of Mod5 (in deep teal color) in the crystal structure of yeast Mod5/tRNACys (PDB: 3EPH). The below the figure is the schematic representation of the domain modularity of Mod5. The catalytic, inserted, and ZnFD of Mod5 are in gray, blue, and deep teal, respectively. A zoom of the superposition on the ZnF motif region is shown on the right. The ZnF motif of Mod5 is indicated below the zoom in the deep teal colored box.

Figure 7. Structural comparison of the ZnFD of Dus2 and that of S. cerevisiae Mod5. ZnFD of S. parasitica Dus2 (in red) is superimposed on the ZnFD of Mod5 (in deep teal color) in the crystal structure of yeast Mod5/tRNACys (PDB: 3EPH). The below the figure is the schematic representation of the domain modularity of Mod5. The catalytic, inserted, and ZnFD of Mod5 are in gray, blue, and deep teal, respectively. A zoom of the superposition on the ZnF motif region is shown on the right. The ZnF motif of Mod5 is indicated below the zoom in the deep teal colored box.

Figure 8. Structural analysis of various Dus2 domains. (A) Stereoview of the structural superimposition of the crystal structure of the dsRBD of A. queenslandica Dus2 (colored in pink) with the dsRBD obtained from the 3D model of A. queenslandica Dus2 (colored in red) generated by AlphaFold2. The side chains are shown as lines. (B) Stereoview of the structural superimposition of the crystal structure of the dsRBD of A. queenslandica Dus2 (colored in pink) with the crystal structure of the hDus2 dsRBD (colored in deep teal) in complex with a dsRNA (PDB: 5OC6). The backbone of the dsRNA is orange, while the nucleosides are deep teal. (C) Structural superimposition of the PyrOx domain of M. pennsylvanicum from the Dus2 model with the X-ray structure of the dimer of E. coli pyridoxine 5′-phosphate oxidase complexed with pyridoxal 5′-phosphate (PLP) and flavin mononucleotide (FMN) (PDB: 1G79). The dimer of PyrOx generates two equivalent active sites, each containing a PLP and an FMN. PyrOx of Dus2 and 1G79 are in red and purple, respectively. The FMN and PLP represented as sticks are in yellow and purple, respectively. (D) Electrostatic surface of PyrOx domain of M. pennsylvanicum Dus2. (E) Structural superimposition of the KPHMT domain of P. insidiosum from the Dus2 model (in red) with the crystal structure of ketopantoate hydroxymethyltransferase complexed the product ketopantoate (PDB: 1M3U, colored in violet). (F) Electrostatic surface of the KPHMT domain of P. insidiosum from the Dus2 model. (G) Electrostatic surface of Dus2 model from T. nelsoni.

Figure 8. Structural analysis of various Dus2 domains. (A) Stereoview of the structural superimposition of the crystal structure of the dsRBD of A. queenslandica Dus2 (colored in pink) with the dsRBD obtained from the 3D model of A. queenslandica Dus2 (colored in red) generated by AlphaFold2. The side chains are shown as lines. (B) Stereoview of the structural superimposition of the crystal structure of the dsRBD of A. queenslandica Dus2 (colored in pink) with the crystal structure of the hDus2 dsRBD (colored in deep teal) in complex with a dsRNA (PDB: 5OC6). The backbone of the dsRNA is orange, while the nucleosides are deep teal. (C) Structural superimposition of the PyrOx domain of M. pennsylvanicum from the Dus2 model with the X-ray structure of the dimer of E. coli pyridoxine 5′-phosphate oxidase complexed with pyridoxal 5′-phosphate (PLP) and flavin mononucleotide (FMN) (PDB: 1G79). The dimer of PyrOx generates two equivalent active sites, each containing a PLP and an FMN. PyrOx of Dus2 and 1G79 are in red and purple, respectively. The FMN and PLP represented as sticks are in yellow and purple, respectively. (D) Electrostatic surface of PyrOx domain of M. pennsylvanicum Dus2. (E) Structural superimposition of the KPHMT domain of P. insidiosum from the Dus2 model (in red) with the crystal structure of ketopantoate hydroxymethyltransferase complexed the product ketopantoate (PDB: 1M3U, colored in violet). (F) Electrostatic surface of the KPHMT domain of P. insidiosum from the Dus2 model. (G) Electrostatic surface of Dus2 model from T. nelsoni.

Figure 9. Evolution of tRNA binding mode in Dus enzymes catalyzing D20 biosynthesis. (A) Crystal structure of T. thermophilus DusA in complex with tRNA (PDB: 3B0V). (B,C) Structural models of S. cerevisiae Dus2/tRNA and hDus2/tRNA complexes, respectively. The TIM-barrel domain (TBD) appears in teal, while the helical domain (HD) is in blue, the inserted beta-sheet in red, the connecting c-αH in green, and the dsRBD in purple. The FMN coenzyme is denoted in yellow. The electrostatic surface of each Dus protein is shown below the protein or tRNA.

Figure 9. Evolution of tRNA binding mode in Dus enzymes catalyzing D20 biosynthesis. (A) Crystal structure of T. thermophilus DusA in complex with tRNA (PDB: 3B0V). (B,C) Structural models of S. cerevisiae Dus2/tRNA and hDus2/tRNA complexes, respectively. The TIM-barrel domain (TBD) appears in teal, while the helical domain (HD) is in blue, the inserted beta-sheet in red, the connecting c-αH in green, and the dsRBD in purple. The FMN coenzyme is denoted in yellow. The electrostatic surface of each Dus protein is shown below the protein or tRNA.

Table 1. X-ray structures of Dus available in the protein data bank.

Table 1. X-ray structures of Dus available in the protein data bank.

ProteinsProductsDomain/ComplexPDB CodeResolutionT. thermophilus DusAD20/D20afull length3B0P1.7T. thermophilus DusAD20/D20afull length + RNA fragment3B0U1.94T. thermophilus DusAD20/D20afull length + tRNAPhe3B0V3.51E. coli DusBD17full length6EI92.55E. coli DusCD16full length3W9Z2.1E. coli DusCD16full length4BFA1.65E. coli DusCD16full length + tRNATrp4YCP2.55E. coli DusCD16full length + tRNAPhe4YCO2.1Homo sapiens Dus2D20TIM Barrel + HD4XP71.9Homo sapiens Dus2D20TIM Barrel + HD4WFS2.68Homo sapiens Dus2D20dsRBD4WFT1.7Homo sapiens Dus2D20dsRBD + dsRNA5OC63.2

Table 2. Data collection and refinement statistics of the dsRBD of A. queenslandica Dus2.

Table 2. Data collection and refinement statistics of the dsRBD of A. queenslandica Dus2.

dsRBDaqPDB code8B02Data collection Wavelength (Å)0.9801Resolution range (Å)42.37–1.68 (1.70–1.68)Space groupP21Cell dimensions          a, b, c (Å)29.077, 56.895, 63.587          α, β, γ (°)90.00, 93.05, 90.00Multiplicity2.9 (2.0)Completeness (%)97.9 (79.7)Mean I/sigma(I)7.8 (0.8)Wilson B-factor (Å2)24.07R-meas0.090 (1.245)R-pim0.051 (0.762)CC1/20.997 (0.351)Refinement Reflections used in refinement23356 (1170)R-work / R-free (%)20.35/22.78 (32.80/36.10)Number of non-hydrogen atoms           macromolecules1548          ligands28          solvent139R.m.s. deviations           Bond lengths (Å)0.009          Bond angles (°)1.02Ramachandran plot (%)           favored98.96          allowed1.04          outliers0.00Average B-factor (Å2)           Overall28.45          macromolecules26.91          ligands42.77          solvent42.75