Mutants of 4-Oxalocrotonate Tautomerase Catalyze the Decarboxylation of Oxaloacetate through an Imine Mechanism

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

“…Incubation of the P1S variant (1 mg) with pyruvate (10 mM), however, yielded an ESI‐MS spectrum that showed a major peak at 6802 ± 1 Da (P1S monomer) and a minor peak at 6874 ± 1 Da (P1S monomer + 72 Da) in a ratio of 5–1. These findings are consistent with the observations reported by Dawson and coworkers, the 72 Da adduct corresponding to pyruvate [18]. The reversal of the ratio between the native and the alkylated monomer that was observed when the P1S variant was incubated with pyruvate (instead of oxaloacetate), strongly suggests that the pyruvate‐adduct that is observed when the protein is incubated with oxaloacetate is an intermediate of the decarboxylation reaction, rather than the result of alkylation by pyruvate (as a product of decarboxylation) that is released into solution.…”

“…The oxaloacetate decarboxylase activity of the 4‐OT P1S and 4‐OT P1A variants was measured by following the depletion of the enol‐form of oxaloacetate (oxobutanedioic acid) in 50 mM NaH 2 PO 4 buffer (pH 7.3) at 22 °C [18]. A stock solution (1 M) of oxaloacetate was made by dissolving the crystalline free acid in absolute ethanol directly before use.…”

“…Drawbacks of this method are the high costs involved and the fact that the synthetic protein needs to be reconstituted in buffer in a properly folded and active form. While this is an experimental challenge, it has been demonstrated to work for 4‐OT [10,11,18]. Dawson and coworkers chemically synthesized the P1G and P1A variants of 4‐OT, thereby introducing a primary amine functionality at the amino‐terminus of the enzyme replacing the secondary amine functionality of Pro‐1 [18].…”

“…While this is an experimental challenge, it has been demonstrated to work for 4‐OT [10,11,18]. Dawson and coworkers chemically synthesized the P1G and P1A variants of 4‐OT, thereby introducing a primary amine functionality at the amino‐terminus of the enzyme replacing the secondary amine functionality of Pro‐1 [18]. Interestingly, both the P1G and P1A variants were found to promiscuously catalyze the decarboxylation of oxaloacetate through a covalent imine‐mechanism, yielding pyruvate and carbon dioxide as the products (Fig.…”

“…One way of obtaining Pro-1 variants of 4-OT in a pure and fully demethionylated form is by peptide synthesis [18]. Drawbacks of this method are the high costs involved and the fact that the synthetic protein needs to be reconstituted in buffer in a properly folded and active form.…”