Construction and in vivo assembly of a catalytically proficient and hyperthermostable de novo enzyme

Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H2O2. The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes

Dimerisation of N-acetyl-L-tyrosine ethyl ester and Aß peptides via formation of dityrosine

Alzheimer\u27s disease (AD) is characterised by the formation of amyloid deposits composed primarily of the amyloid &beta;-peptide (A&beta;). This peptide has been shown to bind redox active metals ions such as copper and iron, leading to the production of reactive oxygen species (ROS) and formation of hydrogen peroxide (H2O2). The generation of H2O2 has been linked with A&beta; neurotoxicity and neurodegeneration in AD. Because of the relative stability of a tyrosyl radical, the tyrosine residue (Tyr-10) is believed to be critical to the neurotoxicity of A&beta;. This residue has also been shown to be important to A&beta; aggregation and amyloid formation. It is possible that the formation of an A&beta; tyrosyl radical leads to increased aggregation via the formation of dityrosine as an early aggregation step, which is supported by the identification of dityrosine in amyloid plaque. The role of dityrosine formation in A&beta; aggregation and neurotoxicity is as yet undetermined, partly because there are no facile methods for the synthesis of A&beta; dimers containing dityrosine. Here we report the use of horseradish peroxidase and H2O2 to dimerise N-acetyl-l-tyrosine ethyl ester and apply the optimised conditions for dityrosine formation to fully unprotected A&beta; peptides. We also report a simple fluorescent plate reader method for monitoring A&beta; dimerisation via dityrosine formation. <br /