A robust protein host for anchoring chelating ligands and organocatalysts.
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.
Reetz
Manfred T
MT
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr, Germany. reetz@mpi-muelheim.mpg.de
Rentzsch
Martin
M
Pletsch
Andreas
A
Taglieber
Andreas
A
Hollmann
Frank
F
Mondière
Régis J G
RJ
Dickmann
Norbert
N
Höcker
Birte
B
Cerrone
Simona
S
Haeger
Michaela C
MC
Sterner
Reinhard
R
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
D002614:Chelating Agents; D008024:Ligands; D008301:Maleimides; D011506:Proteins