Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme

Abstract

Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G(734) of pyruvate formate-lyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. The activase performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine (AdoMet), thus placing it among the AdoMet radical superfamily of enzymes. We report here structures of the substrate-free and substrate-bound forms of pyruvate formate-lyase-activating enzyme, the first structures of an AdoMet radical activase. To obtain the substrate-bound structure, we have used a peptide substrate, the 7-mer RVSGYAV, which contains the sequence surrounding G(734). Our structures provide fundamental insights into the interactions between the activase and the G(734) loop of pyruvate formate-lyase and provide a structural basis for direct and stereospecific H atom abstraction from the buried G(734) of pyruvate formate-lyase.

Fig. 1.

Stereoview of PFL-AE with secondary structural elements assigned numerically (helices in cyan, strands in yellow). The loops after strands β1′, β1, and β6 are labeled A (red, residues 10–20), B (purple, residues 27–47), and C (orange, residues 201–225). The 4Fe-4S cluster (ruby and gold), AdoMet (green carbons), and peptide (teal carbons) are depicted in sticks with oxygens colored red and nitrogens colored blue.

Fig. 2.

Substrate and cofactor binding. Colors are as in Fig. 1, with protein side chain carbons in gray. Conserved motifs (33) are labeled in blue. Composite omit maps are shown as a blue mesh and are contoured at 1σ. Hydrogen bond lengths and other distances are represented as dashed lines. (A) Detail of cluster–AdoMet interaction with composite omit map contoured around the AdoMet. Distances of interest between the unique iron of the 4Fe-4S cluster and AdoMet atoms are shown. (B) AdoMet–protein interactions. (C) Peptide–protein interactions. (D) Omit map contoured around the peptide.

Fig. 3.

Conformational change of PFL-AE upon substrate binding. Colors are as in Fig. 1 unless otherwise noted. (A) The AE and pept-AE models were superimposed and colored in gray, with areas undergoing a conformational change highlighted (blue, AE; red, pept-AE). Loops A–C are labeled. (B) Close-up view of the active site showing side-chain rearrangements upon substrate binding. AE carbon atoms are colored blue, pept-AE carbon atoms red, oxygen red, and nitrogen blue.

Fig. 4.

Docking studies of PFL-AE. Colors are as in Fig. 1 unless specified. (A) Surface representation of the pept-AE colored by sequence conservation as calculated by ESPript (43), showing the same view of the pept-AE as in Fig. 1 and rotated 180° from that orientation. Sequence conservation is represented in rainbow colors, with areas of 100% conservation in red, and 0% conservation in blue. The three conserved regions are numbered. (B) Topology diagram of PFL showing 10-stranded α/β barrel (strands in yellow) with RD highlighted in pink and helices denoted 1 (PFL residues 712–720) and 2 (744–752). An N-terminal domain is omitted for clarity. (C) Ribbon representation of the PFL dimer in gray with RD in magenta and G⁷³⁴ in spacefill. (D) Best docking model output by ZDOCK, with C_α of G⁷³⁴ displayed in spacefill and RD colored as in B. (E) Docking model, with RD displayed as in D. The pept-AE is colored as in A and is shown in the same orientation. An arrow indicates a loop from RD. (F) Detail of active site of docking model colored as in D, with side chains of interest shown in sticks. RD side chains are labeled in magenta, pept-AE side chains in black. Dashed lines indicate possible side-chain interactions between RD and AE.