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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  Escherichia   coli  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.

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