title: 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.
authors.affiliation: Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr, Germany. reetz@mpi-muelheim.mpg.de
authors.forename: Manfred T
authors.initials: MT
authors.lastname: Reetz
authors.forename: Martin
authors.initials: M
authors.lastname: Rentzsch
authors.forename: Andreas
authors.initials: A
authors.lastname: Pletsch
authors.forename: Andreas
authors.initials: A
authors.lastname: Taglieber
authors.forename: Frank
authors.initials: F
authors.lastname: Hollmann
authors.forename: Régis J G
authors.initials: RJ
authors.lastname: Mondière
authors.forename: Norbert
authors.initials: N
authors.lastname: Dickmann
authors.forename: Birte
authors.initials: B
authors.lastname: Höcker
authors.forename: Simona
authors.initials: S
authors.lastname: Cerrone
authors.forename: Michaela C
authors.initials: MC
authors.lastname: Haeger
authors.forename: Reinhard
authors.initials: R
authors.lastname: Sterner
mesh_terms: D002384:Catalysis; D002614:Chelating Agents / Q000138:chemical synthesis / Q000737:chemistry*; D015870:Gene Expression; D008024:Ligands; D008301:Maleimides / Q000138:chemical synthesis / Q000737:chemistry; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D002614:Chelating Agents; D008024:Ligands; D008301:Maleimides; D011506:Proteins