A Robust Protein Host for Anchoring Chelating Ligands and Organocatalysts

Abstract

In order to put the previously proposed concept of directed evolution of hybrid catalysts (proteins that harbor synthetic transition-metal catalysts or organocatalysts) into practice, several prerequisites must be met. The availability of a robust host protein that can be expressed in sufficiently large amounts, and that can be purified in a simple manner is crucial. The thermostable enzyme tHisF from Thermotoga maritima, which constitutes the synthase subunit of a bi-enzyme complex that is instrumental in the biosynthesis of histidine, fulfills these requirements. In the present study, fermentation has been miniaturized and parallelized, as has purification of the protein by simple heat treatment. Several mutants with strategically placed cysteines for subsequent bioconjugation have been produced. One of the tHisF mutants, Cys9Ala/Asp11Cys, was subjected to bioconjugation by the introduction of a variety of ligands for potential metal ligation, of a ligand/metal moiety, and of several organocatalytic entities that comprise a flavin or thiazolium salts. Characterization by mass spectrometry and tryptic digestion was achieved. As a result of this study, a platform for performing future directed evolution of these hybrid catalysts is now available.

A General Method for Artificial Metalloenzyme Formation through Strain‐Promoted Azide–Alkyne Cycloaddition

“…[20] Because the bottom of this protein is blocked by a salt-bridge, we hoped that azide or alkyne mutations introduced deep within the 25 Å long central pore of the protein should project SPAAC cofactors up the pore and place the metal complexes within reach of several loops near the pore opening (Scheme 1, Fig. 1A/B, see also supporting information).…”

“…1C), despite A50 and A199 being located on the protein interior [23] . The hexa-histidine tagged scaffold proteins were purified by Ni-affinity chromatography following an initial heat treatment, [20] and both fluorescence (Fig. 1D) and CD spectroscopy (Fig.…”

Manganese terpyridine artificial metalloenzymes for benzylic oxygenation and olefin epoxidation

“…Maleimide-substituted cofactor 3 was synthesized 22,23 from the known amine-substituted terpyridine, 1 24,25 (Scheme 1). …”

“…Both of these proteins have been used as ArM scaffolds by us 28 and by others 29,30 and therefore serve as ideal substrates for initial studies on cofactor bioconjugation using 3 . Reacting tHisF-C48 with 3 provided modest conversion (ca.…”

Bioinspired Catalyst Design and Artificial Metalloenzymes

“…For example, Reetz and co-workers set out to develop a platform for artificial metalloenzymes based on a robust protein specifically chosen for its suitability to develop en masse purification and bioconjugation procedures. [67,115] Also particular protein structures may be rationally matched with the desired (shape) selectivity in a target reaction. However, applications of covalently assembled systems have not been as successful as their supramolecular counterparts, whose straightforward assembly greatly facilitates combinatorial screening efforts.…”