COASY discussion

CoA is an essential cofactor in living cells, synthesised from pantothenate (vitamin B5) by a five step pathway that is shared by both prokaryotes and eukaryotes. In eukaryotes, the final two steps of this pathway are catalysed by a single bifunctional enzyme, CoAsy, and previous research has confirmed that mouse CoAsy is a 563 amino acid protein encoded by the Ukr1 gene, and possessing two domains (DPCK and PPAT).

Initial analysis of CoAsy expression confirmed that the expression of CoAsy is consistent with its function. CoA is involved in widespread metabolic processes (eg. the tricarboxylic acid cycle) and the baseline expression of CoAsy in all tissues facilitates synthesis of the CoA needed in for these processes. However, CoA is also involved in other processes such as fatty acid synthesis, which occurs primarily in the liver and adipose tissue (Fox, 2004, p115). This is consistent with the higher levels of CoAsy expression in these tissues. The reason for the high level of CoAsy expression in the kidney is, however, less clear. Also, while baseline expression of the other enzymes involved in CoA synthesis (pantothenate kinase, phosphopantothenoylcysteine synthase, phosphopantothenolycysteine decarboxylase) was also present in all tissues, their expression was otherwise quite dissimilar to that of CoAsy. This may have been due to use of intermediates of CoA synthesis in other tissues, however, this possibility requires further investigation. Nonetheless, the elevated expression of CoAsy in the adipose tissue, and particularly in the liver, is consistent with its function in CoA synthesis. Similarly, the discovery of Zhyvoloup et. al. (2003) that CoAsy is associated with the mitochondrial outer membrane is consistent with its function in CoA synthesis, as CoA is used in the mitochondria during the tricarboxylic acid cycle (Fox, 2004, p108).

The crystal structure of part of CoAsy has recently been determined, and by analysing this structure we have shed further light on the structural composition and mechanism of action of this essential enzyme.

Bioinformatic analysis revealed that CoAsy chain A contains the DPCK domain of CoA and part of its PPAT domain. The presence of the DPCK domain was reflected in the high structural similarity of CoAsy chain A to several other kinases, including polynucleotide kinase, adenylate kinase, deoxyguanasine kinase and adenosine-5’phosphosulfate kinase. However, most of the PPAT domain was not present on chain A, as it was positioned before residue 295 of the full-length protein, which was removed during structural determination.

However, the whole of the DPCK domain was present on chain A, and putative binding sites for its ligands (ATP and dephospho-CoA) were determined. Strong evidence suggested that ATP binds in the region of the DPCK domain shown in Figure 5. Pattern searches revealed that a P-loop (an ATP/GTP binding motif) was present in this region. It contained S72 and G75, which were predicted ligand binding residues. Moreover, proteins identified as being structurally similar to CoAsy (chain A) showed particularly strong structural similarity in this region (see Figure Five). The MSA shows that the same was true for sequence similarity (see Figure 6). Together, this strongly suggests that ATP binds in the region shown in Figure Three. In future, this could be confirmed by experimental approaches. Dephospho-CoA may bind to the DPCK domain between the region of its 5th and 7th alpha-helices. During structural determination, acetyl-CoA cocrystallised in this region of CoAsy. Because of the similar structure of dephospho-CoA and acetyl-CoA (see Figure 7), we have predicted that this region may correspond to the dephospho-CoA binding site. This is supported by the presence of predicted ligand binding residues in this region (R128, L131, V135, F136, M142, L145, T146, W150, I153). It is also suggested by the similarity of this region in different species, shown on our MSA (see Figure Seven). However, the position of the binding site and the precise residues involved needs to be further confirmed by experimental approaches, ideally using dephospho-CoA rather than acetyl-CoA. This is particularly necessary because predicted ligand binding residues and sequence similarity did exist in regions of the protein that were not predicted to bind ATP or dephospho-CoA.

Because most of the PPAT domain was not present on CoAsy Chain A, binding sites for its ligands (ATP and 4’ phosphopantotheine) could not be determined. These binding sites did not appear to reside on the small part of the PPAT domain that was present, as they were not revealed by pattern searches.

Also, because it did not contain the PPAT domain, chain A could not be used to determine the structural interaction between the PPAT and DPCK domains of CoAsy. Further studies to determine the structure and ligand binding sites of the PPAT domain of CoAsy, together with our predictions of the ligand binding sites on the DPCK domain, could shed valuable light on the interactions between the two domains and the functional significance of these. This may, for example, help to determine whether tunnelling of dephospho-CoA occurs between the PPAT and DPCK domains. This could help resolve the controversy over whether the lack of dephospho-CoA accumulation in cells is due to PPAT being a rate-limiting enzyme, or, as Daugherty et. al. (2002) suggested, to this tunnelling effect. Whilst the domains occur on opposite sides of the protein, the missing string of 282 residues constituting the remainder of the PPAT domain could wrap around the back of the structure and receive dephospho-CoA through the opening on the rear side of the ACO cleft (see Figure 5). This would then allow the DPCK domain to tunnel it’s by product dephospho-CoA to the PPAT domain for the next step in the pathway. As the current structurally determined A strand of CoAsy was not found to contain the binding site for dephospho-CoA on the PPAT domain, it is then quite possible that the missing segment has this binding site and is able to receive dephospho-CoA through this mechanism.

Figure 7

Structures of Dephospho-CoA (A) and Acetyl-CoA (B). Structure of dephospho-CoA was reproduced from Daugherty et. al., 2002. Structure of Acetyl-CoA was reproduced from Baggot & Dennis, 1998.

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Abstract | Introduction | Results | Discussion | Conclusion | Method | References

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