Catalytic promiscuity of 4-oxalocrotonate tautomerase. Discovery and characterization of C-C bond-forming activities

PhD ceremony: Ms. E. Zandvoort, 14.30 uur, Academiegebouw, Broerstraat 5, Groningen

Dissertation: Catalytic promiscuity of 4-oxalocrotonate tautomerase. Discovery and characterization of C-C bond-forming activities

Promotor(s): prof. W.J. Quax, prof. G.J. Poelarends

Faculty: Mathematics and Natural Sciences

A powerful, recently emerged theme that is highly relevant to the design of new enzymes is that of catalytic promiscuity, where an enzyme catalyzes an alternative reaction (or reactions) in addition to its biologically relevant one. The work of Ellen Zandvoort shows that screening for new promiscuous reactions of an enzyme on the basis of its unique characteristics(the presence of a catalytic amino-terminal proline that has the reactivity to form enamines with carbonyl compounds) is a powerful strategy to find new synthetically useful catalytic transformations.

Enzyme promiscuity has great promise as a source of synthetically useful catalytic transformations. The challenge is to use the understanding of reaction mechanisms to discover new promiscuous reactions in existing enzymes, and exploiting this promiscuity to create tailor-made biocatalysts.

The enzyme 4-oxalocrotonate tautomerase (4-OT) naturally catalyzes tautomerization reactions and uses its unique N-terminal proline (pKa~6.4) as catalytic base. Inspired by the success of L-proline in organocatalysis of C-C bond-forming reactions, Zandvoort explored the proline-based tautomerase 4-OT for its ability to catalyze unnatural C-C bond-forming reactions. She found that 4-OT is highly promiscuous and catalyzes C-C bond formation in several aldol (condensation) reactions and a Michael-type addition reaction. Interestingly, the product of the latter reaction is 4-nitro-3-phenyl-butanal, an important precursor for the anti-depressant Phenibut (4-amino-3-phenyl-butanoic acid). In addition, she discovered a promiscuous cis-trans isomerization activity. Characterization of the different activities indicated that the N-terminal proline residue of 4-OT is indeed essential for all of these promiscuous reactions, and catalysis likely takes place through an enamine intermediate.