A Robust Protein Host for Anchoring Chelating Ligands and Organocatalysts

Authors

  • Manfred T. Reetz Prof. Dr.,

    1. Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr, Germany, Fax: (+49) 208-306-2985
    Search for more papers by this author
  • Martin Rentzsch,

    1. Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr, Germany, Fax: (+49) 208-306-2985
    Search for more papers by this author
    • Deceased on 19.05.2004.

  • Andreas Pletsch Dr.,

    1. Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr, Germany, Fax: (+49) 208-306-2985
    Search for more papers by this author
  • Andreas Taglieber Dr.,

    1. Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr, Germany, Fax: (+49) 208-306-2985
    Search for more papers by this author
  • Frank Hollmann Dr.,

    1. Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr, Germany, Fax: (+49) 208-306-2985
    Search for more papers by this author
  • Régis J. G. Mondière Dr.,

    1. Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr, Germany, Fax: (+49) 208-306-2985
    Search for more papers by this author
  • Norbert Dickmann,

    1. Max-Planck-Institut für Bioanorganische Chemie, Stiftstrassee 34-36, 45470 Mülheim/Ruhr, Germany
    Search for more papers by this author
  • Birte Höcker Dr.,

    1. Institut für Biochemie, Universität zu Köln, Otto-Fischer-Strasse 12-14, 50674 Köln, Germany
    Search for more papers by this author
  • Simona Cerrone,

    1. Institut für Biochemie, Universität zu Köln, Otto-Fischer-Strasse 12-14, 50674 Köln, Germany
    Search for more papers by this author
  • Michaela C. Haeger Dr.,

    1. Institut für Biochemie, Universität zu Köln, Otto-Fischer-Strasse 12-14, 50674 Köln, Germany
    2. Institut für Biophysik und physikalische Biochemie, Universität Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
    Search for more papers by this author
  • Reinhard Sterner Prof. Dr.

    1. Institut für Biochemie, Universität zu Köln, Otto-Fischer-Strasse 12-14, 50674 Köln, Germany
    2. Institut für Biophysik und physikalische Biochemie, Universität Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
    Search for more papers by this author

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.

Ancillary