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

ABSTRACT: 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-hy

SUBMITTER: Baas BJ 

PROVIDER: S-EPMC4141196 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature