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A Robust Protein Host for Anchoring Chelating Ligands and Organocatalysts
Author(s) -
Reetz Manfred T.,
Rentzsch Martin,
Pletsch Andreas,
Taglieber Andreas,
Hollmann Frank,
Mondière Régis J. G.,
Dickmann Norbert,
Höcker Birte,
Cerrone Simona,
Haeger Michaela C.,
Sterner Reinhard
Publication year - 2008
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200700413
Subject(s) - bioconjugation , thermotoga maritima , chemistry , combinatorial chemistry , histidine , moiety , ligand (biochemistry) , directed evolution , biochemistry , enzyme , mutant , stereochemistry , escherichia coli , receptor , gene
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.