Structural and functional insights into Saccharomyces cerevisiae riboflavin biosynthesis reductase RIB7

Abstract

Saccharomyces cerevisiae RIB7 (ScRIB7) is a potent target for anti-fungal agents because of its involvement in the riboflavin biosynthesis pathway as a NADPH-dependent reductase. However, the catalytic mechanism of riboflavin biosynthesis reductase (RBSRs) is controversial, and enzyme structure information is still lacking in eukaryotes. Here we report the crystal structure of Saccharomyces cerevisiae RIB7 at 2.10 Å resolution and its complex with NADPH at 2.35 Å resolution. ScRIB7 exists as a stable homodimer, and each subunit consists of nine central β-sheets flanked by five helices, resembling the structure of RIB7 homologues. A conserved G(76)-X-G(78)-Xn-G(181)-G(182) motif is present at the NADPH pyrophosphate group binding site. Activity assays confirmed the necessity of Thr79, Asp83, Glu180 and Gly182 for the activity of ScRIB7. Substrate preference of ScRIB7 was altered by mutating one residue (Thr35) to a Lysine, implying that ScRIB7 Thr35 and its corresponding residue, a lysine in bacteria, are important in substrate-specific recognition.

Figure 1. Overall structure of ScRIB7 and its NADPH binary complex.

A, ScRIB7 homodimer. The dimer is viewed along the noncrystallographic 2-fold axis. Molecules A and B are colored in green and cyan, respectively. B, ScRIB7 monomer. Secondary structures are numbered according to the primary sequence. C, ScRIB7-NADPH binary complex. An additional helix α2′ is formed upon NADPH cofactor binding. D, Surface electrostatic distribution of the ScRIB7-NADPH binary complex. Positively charged residues are in blue, negatively charged residues are in red and neutral residues are in white. NADPH is shown as sticks: carbon (pink), oxygen (red), nitrogen (blue).

Figure 2. The structure superposition of ScRIB7-NADPH binary complex with homolog structures.

ScRIB7-NADPH chain A is superposed with the cofactor binding structures of MjRIB7 (2AZN, chain A), EcRibD reductase domain (2O7P, chain A) and BsRibG reductase domain (2D5N, chain B). ScRIB7 is shown in green, NADPH bound to ScRIB7 is shown as yellow sticks, MjRIB7 is shown in limon, EcRibD is shown in blue, BsRibG is shown in cyan. The overall structure of ScRIB7 and available homolog structures are very similar, still some significant differences are present. ScRIB7 has a unique α1 compared to MjRIB7, while EcRibD and BsRibG have N-terminal deaminase domains (not shown). Lβ4–β5 is similar to the loop in EcRibD; while the corresponding residues formed α-helices in MjRIB7 and BsRibG (Lβ4–β5 is invisible for it locates inside the helices of MjRIB7 and BsRibG). Lβ5-α4 is longer than those in available homolog structures while Lβ7–β8 is shorter. The substrate binding site between α2 and α2′ is schematically shown as a red dotted circle.

Figure 3. Sedimentation velocity analysis of ScRIB7.

A, The raw sedimentation signals acquired at different time points. Protein sample was prepared as OD₂₈₀ absorbance about 0.8 in buffer containing 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. The sample was scanned at intervals of 30 s for 4 h. (b) Continuous molar mass distribution of the protein showing a single peak with a molecular mass of ∼43 kDa, which is closest to the mass of a ScRIB7 dimer.

Figure 4. The cofactor binding site of ScRIB7 chain A.

Residues of ScRIB7 that are within 4 Å around the cofactor and Asp83 are shown. Both ScRIB7 and the cofactor NADPH are shown as sticks: carbon from ScRIB7 (green), carbon from NADPH (yellow), oxygen (red), and nitrogen (blue).

Figure 5. Multiple-sequence alignment of ScRIB7 with RBSRs from different organisms.

The multisequence alignment was performed using ClustalW2 and ESPript , . Secondary structural elements of ScRIB7 are displayed above the sequence. All sequences were downloaded from the ExPASy database (UniProt accession numbers in parentheses): S. cerevisiae RIB7 (P33312), A. gossypii RIB7 (Q757H6), C. glabrata RIB7 (Q6FU96), K. marxianus RIB7 (Q9P4B8), D. hansenii RIB7 (Q6BII9), M. jannaschii Rib7 (Q58085), M. thermautotrophicus RIB7 (O26337), A. fulgidus RIB7 (O28272), B. subtilis RibG (P17618), E. coli RibD (P25539), S. aureus RibD (D0K610). Filled blue rectangle: key residues involved in substrate-specific recognition; filled black stars: key residues that may play key roles in reduction catalysis; filled green triangle: glycines of G⁷⁶-X-G⁷⁸-X_n-G¹⁸¹-G¹⁸² motif. An additional helix α2′ is formed upon NADPH cofactor binding. Sequences are not shown in integrity.

Figure 6. Enzyme activity assays of ScRIB7 mutants.

A, Relative reductase activity of ScRIB7 mutants toward DAROPP. B&C, Relative reductive activity of T35K mutant and wild type ScRIB7 toward DAROPP or AROPP as substrates. DAROPP and AROPP molecular structures are shown in the inset. Results are presented as means ± S.D. of three independent experiments.

Figure 7. Results from ITC assays of ScRIB7 enzymes.

Interactions between enzymes and NADPH were detected by isothermal titration calorimetry using nano-ITC2g. The protein was dialyzed into PBS and diluted to 80 µM. It was then titrated by 800 µM NADPH dissolved in the same buffer (10 µl NADPH was injected every 300 s). The interaction heat flow was monitored to calculate their affinity. Results were analyzed with NanoAnalyze (TA Instruments). A&B, WT and D83A mutant binding curves obtained from plots of the heat from each NADPH injection against injections over time. C, Spikes of heat flow observed when T79A mutant was titrated by NADPH. D, Calculated best fit parameters.