Exploring routes to stabilize a cationic pyridoxamine in an artificial transaminase: site-directed mutagenesis versus synthetic cofactors.

Two artificial transaminases were assembled by linking a pyridoxamine derivative within an engineered fatty acid binding protein. The goal of mimicking a native transamination site by stabilizing a cationic pyridoxamine ring system was approached using two different strategies. First, the scaffold of intestinal fatty acid binding protein (IFABP) was tailored by molecular modeling and site-directed mutagenesis to position a carboxylate group close to the pyridine nitrogen of the cofactor. When these IFABP mutants (IFABP-V60C/L38K/E93E and -V60C/E51K/E93E) proved to be unstable, a second approach was explored. By N-methylation of the pyridoxamine, a cationic cofactor was created and tethered to Cys60 of IFABP-V60C/L38K and -V60C/E51K; this latter strategy had the effect of permanently installing a positive charge on the cofactor. These chemogenetic assemblies catalyze the transamination between alpha-ketoglutarate and various amino acids with enantioselectivities of up to 96% ee. The pH profile of the initial rates is bell shaped and similar to native aminotransferases. The k(cat) values and the turnover numbers for these new constructs are the highest achieved to date in our system. This success was only made possible by the unique flexibility of the underlying enzyme design concept employed, which permits full control of both the protein scaffold and the catalytically active group.

Semisynthetic Enzymes in Asymmetric Synthesis: Enantioselective Reduction of Racemic Hydroperoxides Catalyzed by Seleno-Subtilisin.

The serine protease subtilisin was chemically converted into the peroxidase-active seleno-subtilisin. This semisynthetic enzyme catalyzes the enantioselective reduction of racemic hydroperoxides in the presence of thiophenols to yield optically active hydroperoxides and alcohols on the semipreparative scale. The kinetic parameters and enantioselectivities of seleno-subtilisin-catalyzed reduction of various chiral hydroperoxides were determined. The catalytic efficiency of this semisynthetic enzyme is comparable to that of the native horseradish peroxidase. The sense in the enantioselectivity of the seleno-subtilisin is opposite to the natural enzymes previously used in the synthesis of optically active hydroperoxides. Consequently, the semisynthetic enzyme seleno-subtilisin complements the naturally available peroxidases for the asymmetric synthesis of both enantiomers.

Novel Biocatalysts by Chemical Modification of Known Enzymes: Cross-Linked Microcrystals of the Semisynthetic Peroxidase Seleno-Subtilisin.

The linkage of lysine residues on the surfaces of subtilisin crystals (NH2 -Enz; see Scheme) with glutardialdehyde affords an immobilized biocatalyst of high stability and purity. The replacement of the serine OH group in the active site (Enz-OH) by SeO2 H leads to new activity as a peroxidase. Thus for the first time, chemical enzyme engineering has resulted in a biocatalyst with a modified peptide framework as well as a new catalytically active site. This methodology combines reasonable substrate selectivity of a semisynthetic enzyme with the exceptional stability of cross-linked enzyme crystals.