Demethionylation of Pro-1 variants of 4-oxalocrotonate tautomerase in Escherichia coli by co-expression with an engineered methionine aminopeptidase

•The P1S variant of 4-OT was fully demethionylated by wild-type MetAP.•The P1H and P1Q variants of 4-OT were partially demethionylated by MetAP-∗TG.•Gln-1 is converted into pyro-Glu (pE) by spontaneous intramolecular cyclization.•Replacement of Pro-1 by Gln is a unique method to incorporate pE-1 in 4-OT.

4-Oxalocrotonate tautomerase (4-OT) catalyzes the enol-keto tautomerization of 2-hydroxymuconate, utilizing its N-terminal proline (Pro-1) as general base catalyst. Substituting Pro-1 with bulky or charged residues will result in poor or no post-translational removal of the translation-initiating methionine by the methionine aminopeptidase (MetAP) of the Escherichiacoli expression host. Here, we set out to investigate whether co-expression with previously engineered aminopeptidase MetAP-∗TG can be used to produce the P1S, P1H and P1Q variants of 4-OT in a demethionylated form. The P1S variant, which carries a small residue at the penultimate position (the first position after the initiating methionine), was found to be fully processed by wild-type MetAP. The P1S variant has low-level 2-hydroxymuconate tautomerase and promiscuous oxaloacetate decarboxylase activity. The P1Q and P1H variants of 4-OT, which carry bulky residues at the penultimate position, could only be obtained in a demethionylated form (a minor fraction of the purified protein is still composed of methionylated enzyme) by co-expression with MetAP-∗TG. Interestingly, the Gln-1 residue of the demethionylated P1Q variant undergoes intramolecular cyclization to form pyroglutamate (pE), yielding variant P1pE. Whereas the P1H/M1P2H mixture has low-level tautomerase activity, the P1pE/M1P2Q mixture has robust tautomerase activity. The substitution of Pro-1 by Gln, followed by removal of the initiating Met and cyclization of Gln-1 to form pE, is a unique way to obtain a structural analogue of proline on the N-terminus of 4-OT. This opens up new possibilities to study the importance of Pro-1 in recently discovered C–C bond-forming activities of this highly promiscuous tautomerase.

Graphical abstractAn amino-terminal proline analogue: The enzyme 4-oxalocrotonate tautomerase (4-OT) is characterized by a catalytic N-terminal proline. The substitution of Pro-1 by a Gln, followed by in vivo removal of the initiating methionine by an engineered methionine aminopeptidase and cyclization of Gln-1 to form pyroglutamate, is a unique way to obtain a structural analogue of proline on the N-terminus of 4-OT.Figure optionsDownload full-size imageDownload as PowerPoint slide