Much Ado About AdoMet

The paper can be accessed at this link:https://onlinelibrary.wiley.com/doi/full/10.1002/pro.3529

QueE is a 7‐Carboxy‐7‐deazaguanine synthase that catalyzes the radical-mediated ring contraction of 6‐carboxy‐5,6,7,8‐tetrahydropterin, forming the pyrrolopyrimidine core of 7‐deazaguanine . QueE is a member of the S‐adenosyl‐L‐methionine (AdoMet), a structurally divergent radical enzyme superfamily, which harnesses the reactivity of radical intermediates to perform its chemical reactions. Members of the AdoMet radical enzyme superfamily utilize a canonical binding motif, a CX3CXϕC 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 (Grell et al. 2019).

Figure 1. Radical S-adenosyl-l-methionine (SAM) enzymes are widely distributed and catalyze diverse reactions. SAM binds to the unique iron atom of a site-differentiated [4Fe-4S] cluster and is reductively cleaved to generate a 5′-deoxyadenosyl radical, which initiates turnover. 7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes a key step in the biosynthesis of 7-deazapurine containing natural products.

The AdoMet radical enzymes harness the cleavage and reduction of a molecule of AdoMet ligated with a [4Fe‐4S] cluster to initiate radical chemistry, requiring a change in the resting oxidation state from +2 to +1. The intermediate generated, 5′‐deoxyadenosyl radical (5′‐dAdo•), is highly reactive and can abstract a hydrogen‐atom (H+) from many substrates, thus enabling many chemically challenging and reactions which can be generated from the reductive cleavage of SAM.

The biological reductant, flavodoxin, was first shown to be capable of this reduction in studies of pyruvate formate‐lyase activating enzyme. Flavodoxin reduces the AdoMet radical cluster as follows; firstly, radical chemistry is initiated through reductive cleavage of AdoMet since the AdoMet radical cluster needs to be reduced from the resting +2 oxidation state to the +1 oxidation state. Finally, low potentials electrons from NADPH are transferred to the AdoMet radical cluster through the action of Ferredoxin, or flavodoxin, an NADP+ reductase mechanism. The need to understand the protein–protein interactions occurring between AdoMet radical enzymes and flavodoxins are integral for exploring the determinants for activation.

The impact of this paper arises from the breadth of knowledge surrounding the catalytic activity of the enzyme, while the design for interaction with physiological reductants remains unclear. This paper examined structural differences between three 7‐carboxy‐7‐deazaguanine synthases and how their differences may be related to the interaction between these enzymes and their biological reductant, flavodoxin.

As we learned in BCM44, the flavin mononucleotide (FMN) cofactor of flavodoxin must be within electron transfer distance from the AdoMet radical cluster for cluster reduction. Therefore, the authors must consider how changes in protein folding observed in these QueE structures could explain the reductant specificity noted above for QueE enzymes. We have also learned that the N‐ and C‐terminal extensions are important for both substrate binding and dimerization in Bm

The authors present the structure of EcQueE i(n the absence of substrate) and compared this structure with previous QueE structures from Bm and Bs. Interestingly, these three QueEs, which all catalyze the exact same reaction, are farther apart in sequence space than are other AdoMet radical enzymes that catalyze completely different reaction

This paper concludes with not only a discussion but also a conclusion, posing a question that they had at the very beginning. By determining the structural data, they were able to evaluate the relationship between fold variation and AdoMet binding, substrate binding, Mg2+ ion binding, and flavodoxin binding, and propose that the QueE structural variation is most likely in response to flavodoxin variations.

5 thoughts on “Much Ado About AdoMet

  1. Thanks for the post, Alyssa! I thought it was interesting that the authors note that interactions between reductants and enzymes are less well-understood that catalytic activity of enzymes. So, you say ” Interestingly, these three QueEs, which all catalyze the exact same reaction, are farther apart in sequence space than are other AdoMet radical enzymes …” What is the relevance/implications of the position of QueEs in the DNA sequence?

  2. Interesting article to say the least. Now if AdoMet radical enzyme induces a change in the 4Fe-4S cluster from +2 to +1 then does it also include a method to prevent radicalizing into a none functional state? Without such a technique would it not be possible for the enzyme to become stuck in one position of its reaction causing problems down the line preventing proper motif binding and formation?

  3. Hi Alyssa, I think this post was unique and interesting to read as the majority of our class has chosen papers with more of a direct therapeutic lens. The idea of ‘co-evolution’ of flavodoxin-QueE pairs is particularly fascinating to me. Given that the three QueE homologs are small and only contain 210-243 amino acids, why and how do you think structure and sequence of the homologs changed over time? I believe the authors credit this to the evolution of flavodoxin, but what could possibly be driving this change?

  4. Thanks Alyssa this paper really challenged me. This research definitely has future work ahead to determine why these enzymes have such differences in structures, but yet catalyze the same reaction. Especially when the authors note other enzymes of this family that catalyze different reactions yet have much less variation in structure among them. Since flavodoxin is hypothesized to drive structural differences, it seems this evolution may have been driven by the failure of this molecule to bind close enough to the 4Fe-4S cluster in these species and therefore failed electron transfer. Did figure 5 also describe some differences in protein structure surrounding the binding of the 4Fe-4S cluster?

  5. I think it is very interesting that the structure of the three homologs are so different but they all catalyze the same reaction. Since their structures are different, Bs and Ec QueE don’t have Mg bound, how they catalyze substrate in each reaction can be different, too (maybe the paper talked about but I missed it). Solving the enzyme structure can the very beginning of the research but it is essential and provides the basis of future study.

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