Epoxide Hydrolases and their Application in Organic Synthesis

The ESTHER database, which is freely available via a web server (http://bioweb.ensam.inra.fr/esther) and is widely used, is dedicated to proteins with an α/β-hydrolase fold, and it currently contains >30 000 manually curated proteins. Herein, we report those substantial changes towards improvement that we have made to improve ESTHER during the past 8 years since our 2004 update. In particular, we generated 87 new families and increased the coverage of the UniProt Knowledgebase (UniProtKB). We also renewed the ESTHER website and added new visualization tools, such as the Overall Table and the Family Tree. We also address two topics of particular interest to the ESTHER users. First, we explain how the different enzyme classifications (bacterial lipases, peptidases, carboxylesterases) used by different communities of users are combined in ESTHER. Second, we discuss how variations of core architecture or in predicted active site residues result in a more precise clustering of families, and whether this strategy provides trustable hints to identify enzyme-like proteins with no catalytic activity. Show more

Our previous contribution to increasing the efficiency of directed evolution is iterative saturation mutagenesis (ISM) as a systematic means of generating focused libraries for the control of substrate acceptance, enantioselectivity, or thermostability of enzymes. We have now introduced a crucial element to knowledge-guided targeted mutagenesis in general that helps to solve the numbers problem in directed evolution. We show that the choice of the amino acid (aa) alphabet, as specified by the utilized codon degeneracy, provides the experimenter with a powerful tool in designing "smarter" randomized libraries that require considerably less screening effort. A systematic comparison of two different codon degeneracies was made by examining the relative quality of the identically sized enzyme libraries in relation to the degree of oversampling required in the screening process. The specific example in our case study concerns the conventional NNK codon degeneracy (32 codons/20 aa) versus NDT (12 codons/12 aa). The model reaction is the hydrolytic kinetic resolution of a chiral trans-disubstituted epoxide, catalyzed by the epoxide hydrolase from Aspergillus niger. The NDT library proves to be of much higher quality, as measured by the dramatically higher frequency of positive variants and by the magnitude of catalyst improvement (enhanced rate and enantioselectivity). We provide a statistical analysis that constitutes a useful guide for the optimal design and generation of "smarter" focused libraries. This type of approach accelerates the process of laboratory evolution considerably and can be expected to be broadly applicable when engineering functional proteins in general. Show more