(493c) GDH to Adh: Complete Redesign of Glucose Dehydrogenase to Alcohol Dehydrogenase

Authors: 
Stahl, L., University of Stuttgart
Bommarius, B., Georgia Institute of Technology
Bommarius, A. S., Georgia Institute of Technology
Pleiss, J., University of Stuttgart

Biocatalysts are increasingly used in industry to create enantiomerically

pure compounds; routes employing dehydrogenases have been especially

successful. Glucose dehydrogenase belongs to the extensive superfamily

of SDRs (short chain dehydrogenase/reductase) and is currently a favored

enzyme for cofactor regeneration. Glucose is its only substrate at a

reasonable rate and the reaction is favored towards oxidation of glucose.

A thermostable glucose dehydrogenase, previously developed at GeorgiaTech, was submitted to several rounds of site-directed mutagenesis, supported by database analysis and structure-guided design, to create an alcohol dehydrogenase based on a glucose dehydrogenase scaffold with a markedly changed substrate specificity. Key to the identification of the necessary amino acid hot spots within the sequence was a bioinformatics approach that involved an extensive data base analysis of existing SDRs

using specific algorithms.  The resulting 3-4 amino acid exchanges led to

a complete change of substrate specificity away from glucose but

respectable activity towards substrates such as cyclopentanol and

1,3-cyclohexandiol. By achieving the goal of de novo design of a glucose

dehydrogenase, we demonstrate the successful implementation of

bioinformatic tools and modeling that can accelerate development of novel

substrate specificity in an otherwise limited enzyme system.