Radical-mediated ring contraction in the biosynthesis of 7-deazapurines

Abstract

Pyrrolopyrimidine containing natural products are widely distributed in Nature. The biosynthesis of the 7-deazapurine moiety that is common to all pyrrolopyrimidines entails multiple steps, one of which is a complex radical-mediated ring contraction reaction catalyzed by CDG synthase. Herein we review the biosynthetic pathways of deazapurines, focusing on the biochemical and structural insights into CDG synthase.

Figure 1

Labeling patterns observed in the biosynthesis of folic acid and deazapurines. In both cases, C-2 (red sphere) of the starting purine is retained but C-8 (grey sphere) is lost. In addition, C-1’, C-2’, and C-3’ (blue spheres) of the proffered purine ribose become incorporated into the final product.

Figure 2

Core steps in the biosynthesis of 7-deazapurines are catalyzed by the successive actions of three enzymes.

Figure 3

Tailoring steps from CDG to (A) the hypermodified tRNA base queuosine and (B) the secondary metabolites toyocamycin and sangivamycin.

Figure 4

Activation of RS enzymes requires reduction of the [4Fe-4S] cluster to the +1 oxidation state. In vivo, NADPH is thought to supply the necessary reducing equivalents via Fpr/Fld (A). Once reduced, the RS cluster catalyzes the reductive cleavage of SAM to generate a dAdo•, which initiates catalysis by H-atom abstraction from the substrate (B).

Figure 5

Reductive activation of B. subtilis CDG synthase with Fld homologs from E. coli and B. subtilis. Both the biological reducing system NADPH/Fld/Fpr (A) and dithionite/Fld (B) are able to activate CDG synthase. Of the Fld homologs, YkuN is able to maintain activity of CDG synthase at significantly lower concentrations than YkuP or Fld.

Figure 6

Mechanism of CDG synthase. Radical-mediated ring contraction is initiated by H-atom abstraction at C-6 (hydrogen in light blue) of the substrate, and product is formed by stereoselective proton abstraction from a putative gem-aminocarboxylate intermediate. Lower inserts show active sites from the structures of CPH₄ (PDB:4NJI), 6-CP (PDB:4NJG), and CDG (PDB: 4NJK) bound near the [4Fe-4S] of CDG synthase. The structure below the gem-aminocarboxylate intermediate is a model based on how CDG binds to CDG synthase. Glu 116 is also shown in this panel. Colors for the structures: Fe in rust, S in yellow, C in green, N in blue, O in red, and modeled H in white. The 7-proS and 7-proR hydrogens are shown in black and red spheres, respectively.

Figure 7

CPH₄, CDG and 6-CP are all bound in the same manner with respect to the SAM-bound [4Fe-4S] cluster in the active site of CDG synthase. Color of the FeS cluster and SAM are as described as in Fig. 6.

Figure 8

Magnesium divalent cation (orange sphere) serves as a major binding determinant for CPH₄ in the active site of CDG synthase. The substrate makes three contacts to the Mg²⁺. A Thr sidechain from the protein is also a ligand. The C-terminal carboxylate and Arg27 are also involved in binding the substrate. Colors are as described in Fig. 6 with the hydrogen that is abstracted (white) modeled into the structure.