Targeted mutagenesis increases the activity and alters the regioselectivity of flavin-dependent halogenases.

Flavin-dependent halogenase (Fl-Hal) enzymes have been shown to halogenate a range of synthetic as well as natural aromatic compounds. The exquisite regioselectively of Fl-Hal enzymes can provide halogenated building blocks which are inaccessible using standard halogenation chemistries. Consequently, Fl-Hal are potentially useful biocatalysts for the chemoenzymatic synthesis of pharmaceuticals and other valuable products, which are derived from haloaromatic precursors. However, the application of Fl-Hal enzymes, in vitro, has been hampered by their poor catalytic activity and lack of stability. To overcome these issues, we identified a thermophilic tryptophan halogenase (Th-Hal), which has significantly improved catalytic activity and stability, compared with other Fl-Hal characterised to date. When used in combination with a thermostable flavin reductase, Th-Hal can efficiently halogenate a number of aromatic substrates. X-ray crystal structures of Th-Hal, and the reductase partner (Th-Fre), provide insights into the factors that contribute to enzyme stability, which could guide the discovery and engineering of more robust and productive halogenase biocatalysts.

The development of a novel method for functionalizing nanopatterned surfaces with catalytically active proteins is reported. This method involves using dip-pen nanolithography (DPN) and polymer pen lithography (PPL) to generate nanoscale patterns of coenzyme A, followed by a phosphopantetheinyl transferase-mediated coupling between coenzyme A and proteins fused to the ybbR-tag. By exploiting the ability to generate protein features over large areas afforded by DPN and PPL, it was now possible to measure protein activity directly on these surfaces. It was found that proteins immobilized on the nanoscale features not only display higher activity per area with decreasing feature size, but are also robust and can be used for repeated catalytic cycles. The immobilization method is applicable to a variety of proteins and gives rise to superior activity compared to proteins attached in random orientations on the surface.

No abstract