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Contenido archivado el 2022-12-23

Structural and mechanistic studies of NAD-dependent formate dehydrogenase

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Based on the X-ray structure of the bacterial formate dehydrogenase (FDH), recently solved by the partners, further work was carried out to refine and interpret the structural data, to strengthen conclusions about the participation of certain amino acids in the catalytic cycle and binding interactions were carried out site directed mutagenesis of the key residues in formate dehydrogenase active centre The mutant proteins were expressed in Escherichia coli and characterized by kinetic and spectroscopic analysis, crystallization of selected muteins is in progress. Besides computer modeling efforts were undertaken to crystallize other NAD+ dependent FDH from the methylotropic yeast Hansenula polymorpha and Candida boidinii to expand the data base. Unfortunately, the handling and crystallization of the yeast enzymes turned out to be more complicated than the bacterial enzyme and crystals suitable for X-ray analysis have not been obtained so far. Key techniques applied are X-ray crystallography, computer based modeling of protein structure, cloning and expression of genes in Escherichia coli, site directed mutagenesis, cultivation of yeast and bacteria in laboratory and pilot scale, isolation and purification of enzymes, enzyme kinetics, spectroscopic analysis of proteins, enzymatic synthesis of chiral alcohols and chiral lactones with NADPH regeneration by a FDH mutant.

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