Crystal Structures of Fsa2 and Phm7 Catalyzing [4 + 2] Cycloaddition Reactions with Reverse Stereoselectivities in Equisetin and Phomasetin Biosynthesis

Abstract

Fsa2 and Phm7 are a unique pair of pericyclases catalyzing [4 + 2] cycloaddition reactions with reverse stereoselectivities in the biosynthesis of equisetin and phomasetin, both of which are potent HIV-1 integrase inhibitors. We here solve the crystal structures of Fsa2 and Phm7, both of which possess unusual "two-β barrel" folds. Different residues are evident between the active sites of Fsa2 and Phm7, and modeling experiments provide key structural information determining the reverse stereoselectivities. These results provide a better understanding of how natural pericyclases control the catalytic stereoselectivities and benefit the protein engineering in future.

Figure 1

[4 + 2] Cycloaddition reactions catalyzed by Fsa2 and Phm7. (A) Fsa2 and Phm7 catalyze the formations of four pairs of reverse chiral centers at C2/C3/C6/C11 in equisetin and phomasetin biosynthesis, respectively. (B) Reactions catalyzed by Fsa2 and Phm7 with reverse stereoselectivities using the same substrate 5. The six-membered rings generated by Fsa2 and Phm7 are labeled in red, and the cycloaddition positions are indicated with blue dashed lines.

Figure 2

Crystal structures and active sites of Fsa2 and Phm7. (A) “Two-β barrel” structure of Fsa2. (B) View from two entrances of the N-terminal β barrel of Fsa2, which is fully filled with nonpolar residues (labeled in yellow sticks). (C) View from two entrances of the C-terminal β barrel of Fsa2, which is fully filled with nonpolar residues (labeled in yellow sticks). (D) “Two-β barrel” structure of Phm7. (E) View from two entrances of the N-terminal β barrel of Phm7, which is fully filled with nonpolar residues (labeled in yellow sticks). (F) View from two entrances of the C-terminal β barrel of Phm7, which is fully filled with nonpolar residues (labeled in yellow sticks). (G) Superimposition of Fsa2 and Phm7 structures. (H) Surface representation of Fsa2 with the same view direction as that for (A). (I) Surface representation of Phm7 with the same view direction as that for (D). The active sites of Fsa2 and Phm7 are indicated with red dashed circles.

Figure 3

Structural comparison of Fsa2 and Phm7 with other pericyclases. (A–D) Different groups of pericyclases based on their structural folds. The α helices and β strands are shown in red and yellow, respectively. The red arrows in (B) indicate the active sites. PDB IDs are shown in parentheses.

Figure 4

Active sites of Fsa2 and Phm7 and modeling experiments. (A) Comparison between active sites of Fsa2 (magenta) and Phm7 (cyan). (B) Modeling of 5 (green) in the active site of Fsa2. (C) Shape complementarities of diene/dienophile moieties of 5 to the active site of Fsa2. Key residues are shown in dot presentation. (D) Modeling of 5 (yellow) in the active site of Phm7. (E) Shape complementarities of diene/dienophile moieties of 5 to the active site of Phm7. Key residues are shown in dot presentation. (F) Superimposition of modeled 5 (green for Fsa2 and yellow for Phm7) in the active sites of Fsa2 and Phm7. Residues’ names are labeled in magenta for Fsa2 and blue for Phm7. The hydrogen bonds are indicated with yellow dashed lines and the cycloaddition positions are indicated with black dashed lines. Red arrows indicate the rotation directions of diene/dienophile moieties during the cycloaddition reactions.