As noted in the RAD001 cell line Introduction, the direct evidence available excludes neither possibility because it is clear that Ygf Z dimerizes readily, at least ex vivo (Teplyakov et al., 2004), and that at least some Ygf Z exists with a free thiol inside plant cells (Hägglund et al., 2008). It will not be possible to show definitively whether Ygf Z works as a disulphide-bonded dimer or as a thiol monomer (or both) until the action of Ygf Z can be reconstituted in vitro. However, the balance of present evidence favours the thiol monomer, as summarized
in the following. Firstly, there is reason to suspect that Ygf Z dimer formation is unphysiological. Thus, the three-dimensional structure of the dimer suggests that the intermolecular C228-C228′ disulphide bridge might not be functionally relevant because the dimer interface formed by multiple nonspecific van der Waals interactions is not extensive and contains none of the conserved dodecapeptide motif residues except C228 (Teplyakov et al., 2004). Moreover, in our pilot tests, recombinant Ygf Z isolated from E. coli was 65% monomeric even when no reductants were added (not shown). Secondly, E. coli Ygf Z has been shown to have a
redox-active cysteine, Doramapimod in vitro i.e. a free thiol group, in vivo (Takanishi et al., 2007). Besides C228, Ygf Z has one other cysteine residue, C63, and it was not shown which is the redox-active one (Takanishi et al., 2007). However, the crystal structure places C63 at the C-terminal end of a β-strand in domain B, which makes the sulfhydryl solvent inaccessible, and C228 in an exposed surface loop between two α-helices (α9 and α10) of the Ygf Z monomer (Teplyakov et al., 2004), suggesting that the latter is the redox-active residue. Finally, Ygf Z belongs to the same protein family as sarcosine oxidase, dimethylglycine oxidase and the T-protein of the glycine-cleavage complex. All of these proteins
use tetrahydrofolate to accept a one-carbon (formaldehyde) unit (Teplyakov et al., 2004; Scrutton & Leys, 2005), and the one structurally closest to Ygf Z – the T-protein – acts on a thiol adduct of the one-carbon unit, borne by the H-protein of the complex (Douce et al., 2001). Formaldehyde is a ubiquitous metabolite that spontaneously forms harmful adducts with reactive protein side chains (Metz et al., DCLK1 2004), and it has been proposed that Ygf Z removes such inhibitory adducts from Fe/S enzymes by transferring the formaldehyde moiety to tetrahydrofolate (Waller et al., 2010). In such an enzyme repair mechanism, a cysteine thiol could logically play a go-between role, analogous to that of the active thiol in the glycine-cleavage complex, by binding formaldehyde after its removal from an Fe/S enzyme and before its transfer to tetrahydrofolate. A repair role for Ygf Z is not incompatible with the proposal that Ygf Z facilitates the breakdown of plumbagin (Lin et al.