Importance of the N-terminal Proline for the Promiscuous Activity of 4-Oxalocrotonate Tautomerase (4-OT)

Abstract

Michael addition of aldehydes to nitroolefins is an effective method to obtain useful chiral γ-nitroaldehydes.  γ-Nitroaldehydes are precursors for chiral γ-aminobytiric acid analogues which have numerous pharmacological activities and are used for treatment of neurological disorders.  A whole-cell system based on recombinantly expressed 4-oxalocrotonate tautomerase (4-OT) has been developed and shown to be an effective biocatalyst for Michael addition of branched aldehydes to β-nitrostyrenes.  The aim of this study was to investigate the influence of the substitution of the N-terminal proline with lysine and arginine both containing the reactive ε-amino group, on the Michael addition catalyzed by 4-OT.  Firstly, the effect of these mutations was examined by in silico analysis, followed by generation of three terminal lysine mutants.  The generated mutants, 4-OT_K, 4-OT_PK and 4-OT_KK were tested for the ability to utilise β-nitrostyrene (1), (E)-2-(thiophene-2-yl)nitroethen (2) and p-chloro-trans-β-nitrostyrene (3) as Michael acceptors with isobutanal as the donor.  For comparison, the lithium salt of lysine was used in the same organocatalytic reactions. In general, the introduction of lysine had a negative effect on Michael additions based on overall product yields.  However, additional lysine residues at the N-terminus of the protein resulted in structural changes that enhanced the activity towards 2 and 3. Therefore, the N-terminal proline is important for the 4-OT catalysed Michael-additons, but it is not essential.