Crystal structure of AdoMet radical enzyme 7-carboxy-7-deazaguanine synthase from Escherichia coli suggests how modifications near [4Fe-4S] cluster engender flavodoxin specificity

Abstract

7-Carboxy-7-deazaguanine synthase, QueE, catalyzes the radical mediated ring contraction of 6-carboxy-5,6,7,8-tetrahydropterin, forming the characteristic pyrrolopyrimidine core of all 7-deazaguanine natural products. QueE is a member of the S-adenosyl-L-methionine (AdoMet) radical enzyme superfamily, which harnesses the reactivity of radical intermediates to perform challenging chemical reactions. Members of the AdoMet radical enzyme superfamily utilize a canonical binding motif, a CX₃ CXϕC motif, to bind a [4Fe-4S] cluster, and a partial (β/α)₆ TIM barrel fold for the arrangement of AdoMet and substrates for catalysis. Although variations to both the cluster-binding motif and the core fold have been observed, visualization of drastic variations in the structure of QueE from Burkholderia multivorans called into question whether a re-haul of the defining characteristics of this superfamily was in order. Surprisingly, the structure of QueE from Bacillus subtilis revealed an architecture more reminiscent of the classical AdoMet radical enzyme. With these two QueE structures revealing varying degrees of alterations to the classical AdoMet fold, a new question arises: what is the purpose of these alterations? Here, we present the structure of a third QueE enzyme from Escherichia coli, which establishes the middle range of the spectrum of variation observed in these homologs. With these three homologs, we compare and contrast the structural architecture and make hypotheses about the role of these structural variations in binding and recognizing the biological reductant, flavodoxin. Broader impact statement: We know more about how enzymes are tailored for catalytic activity than about how enzymes are tailored to react with a physiological reductant. Here, we consider structural differences between three 7-carboxy-7-deazaguanine synthases and how these differences may be related to the interaction between these enzymes and their biological reductant, flavodoxin.

Keywords: AdoMet radical enzymes; flavin mononucleotide; flavodoxin; iron-sulfur clusters; physiological reductant.

Figure 1

Flavodoxin reduces the AdoMet radical cluster. (A) To initiate radical chemistry through reductive cleavage of AdoMet, the AdoMet radical cluster needs to first be reduced from the resting +2 oxidation state to the +1 oxidation state. (B) Low potentials electrons from NADPH are transferred to the AdoMet radical cluster through the action of Ferredoxin (flavodoxin): NADP⁺ reductase/Flavodoxin system. Functional parts of NADPH, FAD and FMN are shown.

Figure 2

Sequence similarity network of the AdoMet radical enzyme subfamily, 7‐carboxy‐7‐deazaguanine synthases (QueE). The protein sequence similarity network38 for the QueE AdoMet radical subfamily was obtained from the Structure Function Linkage Database (http://sfld.rbvi.ucsf.edu/django) and visualized in Cytoscape.39 Each node represents sequences that share 50% identity or higher and node connections are filtered at a Blast Probability of 10⁻²⁵. Nodes are colored based on increasing sequence length; White nodes denote the shortest sequences (149 amino acids) and the orange node denotes the longest sequence (509 amino acids). B. multivorans, B. subtilis and E. coli QueE sequences are shown as diamonds and sequences used in the sequence alignment (Fig. S2) are shown as hexagons and designated with an asterisk (*). QueE catalyzes the AdoMet and magnesium dependent rearrangement of 6‐carboxy‐5,6,7,8‐tetrahydropterin, CPH_4, to 7‐carboxy‐7‐deazaguanine, CDG.

Figure 3

Structure of QueE from Escherichia coli. (A) Structure of EcQueE, shown as ribbons, folds into a head‐to‐tail functional dimer with the dimer interface composed of interactions between the N‐terminal (light pink) and C‐terminal (grey) extensions. The modified AdoMet core, a partial (β₆/α₅) TIM barrel, is shown in blue. (B) EcQueE (blue) monomer overlays well with the monomers of BsQueE, (PDB ID 5TH5), (translucent light green) and BmQueE, (PDB ID 4NJI), (translucent yellow). In both panels, [4Fe–4S] clusters are shown in a ball and stick representation, where iron is colored orange and sulfur is colored yellow.

Figure 4

Topology diagrams for QueE homologs and PFL‐AE. (A) EcQueE, (B) BmQueE, (C) BsQueE and (D) PFL‐AE. The core AdoMet domains are colored blue for EcQueE, yellow for BmQueE and green for BsQueE whereas the N‐ and C‐terminal extensions are colored light pink and grey (respectively) for all three QueE structures. The differences between the QueE homologs structures are shown in bold and the corresponding secondary structure element denoted in magenta and the dashed line delineates the QueE dimer interface. The topology diagram of PFL‐AE is shown with the N‐ and C‐terminal extensions colored pink and slate respectively and AdoMet domain colored in coral. The iron atoms of the [4Fe–4S] clusters are colored orange and sulfur atoms are colored yellow. Cysteine ligands to the [4Fe–4S] cluster are shown as yellow circles. Structural elements outside the AdoMet radical core fold are labeled with a prime.

Figure 5

AdoMet binding pocket in QueE homologs. AdoMet binding within the AdoMet core (translucent ribbons) is shown for (A) BmQueE (PDB ID 4NJI), (B) BsQueE (PDB ID 5TH5) and (C) EcQueE. In (A), AdoMet binding motifs are labeled in magenta. See Fig. S3 for stereo views and further description of AdoMet binding. The binding pockets are composed of residues (sticks), which can provide hydrogen bonds (red) to AdoMet (white). The irons (orange) and the sulfurs (yellow) of the [4Fe‐4S] AdoMet radical cluster are shown as spheres. In (B), the intact AdoMet molecule is modeled using the adenosyl moiety of the 6‐carboxypterin‐5′‐deoxyadenosyl ester adduct (PDB ID 5TH5) and an intact AdoMet molecule (PDB ID 4NJI) as a guide. The AdoMet binding pocket of EcQueE (blue) is shown overlaid with the binding pocket from BmQueE (white) to highlight the changes that need to be made (red arrows) to allow binding of the modeled AdoMet (white) molecule.

Figure 6

Substrate binding pocket. Residues (in sticks) that comprise the substrate‐binding pocket are shown for each of the QueE homologs. (A) CPH₄ is bound to the active site by residues from the N‐terminal extension (pink), the AdoMet radical core fold (yellow) and the C‐terminal extension (grey) of BmQueE (PDB ID 4NJI). (B) In the modeled orientation, CPH₄ appears to interact with the AdoMet radical domain (green) of BsQueE in addition to the N‐ and C‐terminal extensions, colored pink and grey, respectively. (C) CPH₄ modeled into EcQueE. AdoMet radical domain in blue and N‐ and C‐terminal extensions in pink and grey, respectively, are shown overlaid with the active site of BmQueE (white). (D) CPH₄ binding in BmQueE (PDB ID 4NJI) (yellow) creates a magnesium‐binding site. (E) CPH₄ binding in BsQueE (PDB ID 5TH5) (green) is expected to create a magnesium‐binding site similar to that seen in BmQueE. (F) The putative magnesium‐binding site of EcQueE (blue) is shown overlaid with the CPH₄ bound BmQueE (PDB ID 4NJI) (white). The substrate, CPH₄, is shown in lilac, the catalytically essential magnesium is represented as a green sphere, the irons (orange) and the sulfurs (yellow) of the [4Fe–4S] AdoMet radical cluster are shown as spheres, AdoMet is shown in light blue and hydrogen bonds are shown as red dashes. Water molecules (red spheres) necessary for magnesium binding are shown.

Figure 7

Flavodoxins sequence alignment. Sequences include flavodoxins from Helicobacter pylori, Escherichia coli, Anacystis nidulans, Aquifex aeolicus, Desulfovibrio gigas, Clostridium beijerinckii, Streptococcus pneumonia TIGR4, Bacillus subtilis (YkuN), Bacteroides fragilis NCTC 9343, and Burkholderia multivorans. The sequence alignment is colored according to secondary structure, blue for β‐strands and red for α‐helices, and the insertion for long chain flavodoxins and the chain insertion in flavodoxins from B. multivorans and B. fragilis are denoted with a box.

Figure 8

Electrostatic surface charge for EcQueE and the cognate Fld, EcfldA. (A) Ribbon drawing of monomer of EcQueE with the AdoMet radical core in blue and the N‐ and C‐terminals in light pink and grey, respectively, oriented such that the predicted binding sites are facing EcFldA. The structure of EcFldA (PDB ID 1AHN) is also shown in ribbon representation (magenta) with the FMN cofactor and the loops proposed to bind partner proteins facing EcQueE. (B) The solvent accessible electrostatic surface representations of EcQueE and EcFldA with FMN colored salmon are also displayed in the same orientation as in A. Electrostatic potentials are depicted on a colorimetric scale from red to blue for −1 to +1 kTe⁻¹.

Figure 9

QueE orthologs show differential electrostatic surfaces. Ribbon drawing of QueE orthologs, shown as monomers in grey, with the structural elements contributing to the positive electrostatic surface highlighted in cyan. The electrostatic surface potential is shown colored from red to blue for −1 to +1 kTe⁻¹ on the right of each panel for the corresponding orientation of the QueE orthologs. (A) BmQueE, (B) EcQueE, and (C) BsQueE.