Familiar coenzyme in unfamiliar role: The use of formyl-CoA in C1 anabolism

Familiar coenzyme in unfamiliar role: The use of formyl-CoA in C1 anabolism

In 1945, Fritz Lippman discovered the existence of coenzyme A (CoA), which he named after its role in the “activation of acetate” (1). Since then, CoA has been found to be an indispensable molecule, with it and its thioesters estimated to participate in around 4% of enzymatic reactions (2). In recent years we have reported an application of acyl-CoAs in the functional reversal of the β-oxidation pathway, which relies on the use of acetyl-CoA and its derivatives for acyl-chain elongation (3, 4). This work led us to investigate other degradative pathways and their potential for biosynthesis.

A pathway that quickly drew our attention was the variant of the α-oxidation pathway originally discovered in mammals and studied in detail in humans. Like the β-oxidation pathway, the role of the α-oxidation pathway is in fatty acid degradation, and notably, this version of the pathway involves CoA thioester intermediates. Rather than the cleavage of the two-carbon unit acetyl-CoA from the acyl-chain that takes place in β-oxidation, however, in α-oxidation a one-carbon unit is removed in the form of formyl-CoA. We hypothesized that the enzyme catalyzing the key carbon-bond cleavage reaction, 2-hydroxyacyl-CoA lyase (HACL) could act reversibly to catalyze the formation of a carbon-carbon bond between formyl-CoA and a carbon chain and set out to test our hypothesis.

The reaction catalyzed by 2-hydroxyacyl-CoA lyase.
The reaction catalyzed by 2-hydroxyacyl-CoA lyase.

In our study, we report that the HACL-catalyzed reaction is reversible, with formyl-CoA serving as a unit for C1 elongation. Using a TPP-dependent mechanism, HACL was found to condense formyl-CoA with aldehydes, hydroxyaldehydes, and acetone. While CoA is best known for and derives its name from the activation and transfer of acetyl groups, our work shows that the ability for CoA to activate small molecules is extended to C1 formyl groups.

What is interesting, at least to us, is the lack of information in the literature on the participation of formyl-CoA in anabolism. Instead, its physiological role is typically a degradation product rapidly dissimilated to release formate. There is very little evidence, if any, of the use of formyl-CoA for formyl group transfer or for condensation reactions. Why does nature appear to prefer the use of the coenzyme tetrahydrofolate as a C1 carrier and activator, while CoA appears to be preferred for C2 and longer molecules?

We demonstrated that in E. coli the use of formyl-CoA is at least not limited by its relative instability. An engineered strain of E. coli was able to accumulate intracellular formyl-CoA by the oxidation of formaldehyde in sufficient quantity and at timescales relevant for HACL-catalyzed condensation. This not only lays the groundwork for numerous engineered applications of formyl-CoA in biotechnology, but also serves as inspiration for us to investigate whether formyl-CoA has been overlooked. Due to its structural similarity to acetyl-CoA, is it possible that enzymes annotated as having physiological function on acetyl-CoA are actually involved in formyl-CoA metabolism? And perhaps, due to its aforementioned instability, has formyl-CoA evaded detection in metabolomics studies? We hope to shed additional light on these thoughts and provide further examples of the implementations of HACL and formyl-CoA based pathways in future updates.


1.        N. Kresge, R. D. Simoni, R. L. Hill, Fritz Lipmann and the Discovery of Coenzyme A. J. Biol. Chem. 280, 164–167 (2005).

2.        P. K. Mishra, D. G. Drueckhammer, Coenzyme A analogues and derivatives: synthesis and applications as mechanistic probes of coenzyme a ester-utilizing enzymes. Chem. Rev. 100, 3283–3309 (2000).

3.        C. Dellomonaco, J. M. Clomburg, E. N. Miller, R. Gonzalez, Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals. Nature. 476, 355–9 (2011).

4.        S. Cheong, J. M. Clomburg, R. Gonzalez, Energy-and carbon-efficient synthesis of functionalized small molecules in bacteria using non-decarboxylative Claisen condensation reactions. Nat. Biotechnol. 34, 556–561 (2016).

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