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Wageningen, Netherlands

Skalden L.,Institute of Biochemistry | Peters C.,Institute of Biochemistry | Dickerhoff J.,Institute of Biochemistry | Nobili A.,Institute of Biochemistry | And 4 more authors.
ChemBioChem | Year: 2014

Amine transaminases (ATAs) are powerful enzymes for the stereospecific production of chiral amines. However, the synthesis of amines incorporating more than one stereocenter is still a challenge. We developed a cascade synthesis to access optically active 3-alkyl-substituted chiral amines by combining two asymmetric synthesis steps catalyzed by an enoate reductase and ATAs. The ATA wild type from Vibrio fluvialis showed only modest enantioselectivity (14 % de) in the amination of (S)-3-methylcyclohexanone, the product of the enoate-reductase-catalyzed reaction step. However, by protein engineering we created two variants with substantially improved diastereoselectivities: variant Leu56Val exhibited a higher R selectivity (66 % de) whereas the Leu56Ile substitution caused a switch in enantiopreference to furnish the S-configured diastereomer (70 % de). Addition of 30 % DMSO further improved the selectivity and facilitated the synthesis of (1R,3S)-1-amino-3-methylcyclohexane with 89 % de at 87 % conversion. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Vroling B.,Radboud University Nijmegen | Thorne D.,University of Manchester | McDermott P.,University of Manchester | Joosten H.-J.,Bio Prodict | And 3 more authors.
Nucleic Acids Research | Year: 2012

The NucleaRDB is a Molecular Class-Specific Information System that collects, combines, validates and disseminates large amounts of heterogeneous data on nuclear hormone receptors. It contains both experimental and computationally derived data. The data and knowledge present in the NucleaRDB can be accessed using a number of different interactive and programmatic methods and query systems. A nuclear hormone receptor-specific PDF reader interface is available that can integrate the contents of the NucleaRDB with fulltext scientific articles. The NucleaRDB is freely available at http://www.receptors. org/nucleardb. © The Author(s) 2011. Published by Oxford University Press.

Cerdobbel A.,Ghent University | De Winter K.,Ghent University | Aerts D.,Ghent University | Kuipers R.,Radboud University Nijmegen | And 4 more authors.
Protein Engineering, Design and Selection | Year: 2011

Sucrose phosphorylase is a promising biocatalyst for the glycosylation of a wide variety of acceptor molecules, but its low thermostability is a serious drawback for industrial applications. In this work, the stability of the enzyme from Bifidobacterium adolescentis has been significantly improved by a combination of smart and rational mutagenesis. The former consists of substituting the most flexible residues with amino acids that occur more frequently at the corresponding positions in related sequences, while the latter is based on a careful inspection of the enzymes crystal structure to promote electrostatic interactions. In this way, a variant enzyme could be created that contains six mutations and whose half-life at the industrially relevant temperature of 60°C has more than doubled compared with the wild-type enzyme. An increased stability in the presence of organic co-solvents could also be observed, although these effects were most noticeable at low temperatures. © The Author 2011. Published by Oxford University Press. All rights reserved.

Seddon G.,Adelard Institute | Lounnas V.,Radboud University Nijmegen | McGuire R.,Bioaxis Research | Van Den Bergh T.,Bio Prodict | And 3 more authors.
Journal of Computer-Aided Molecular Design | Year: 2012

In its first 25 years JCAMD has been disseminating a large number of techniques aimed at finding better medicines faster. These include genetic algorithms, COMFA, QSAR, structure based techniques, homology modelling, high throughput screening, combichem, and dozens more that were a hype in their time and that now are just a useful addition to the drug-designers toolbox. Despite massive efforts throughout academic and industrial drug design research departments, the number of FDA-approved new molecular entities per year stagnates, and the pharmaceutical industry is reorganising accordingly. The recent spate of industrial consolidations and the concomitant move towards outsourcing of research activities requires better integration of all activities along the chain from bench to bedside. The next 25 years will undoubtedly show a series of translational science activities that are aimed at a better communication between all parties involved, from quantum chemistry to bedside and from academia to industry. This will above all include understanding the underlying biological problem and optimal use of all available data. © 2012 The Author(s).

Aerts D.,Ghent University | Verhaeghe T.,Ghent University | Joosten H.-J.,Bio Prodict | Vriend G.,Radboud University Nijmegen | And 2 more authors.
Biotechnology and Bioengineering | Year: 2013

Consensus engineering, which is replacing amino acids by the most frequently occurring one at their positions in a multiple sequence alignment (MSA), is a known strategy to increase the stability of a protein. The application of this concept to the entire sequence of an enzyme, however, has been tried only a few times mainly because of the problems determining the consensus in highly variable regions. We show that this problem can be solved by replacing such problematic regions by the corresponding sequence of the natural homologue closest to the consensus. When one or a few sub-families are overrepresented in the MSA the consensus sequence is a biased representation of the sequence space. We examine the influence of this bias by constructing three consensus sequences using different MSAs of sucrose phosphorylase (SP). Each consensus enzyme contained about 70 mutations compared to its closest natural homologue and folded correctly and displayed activity on sucrose. Correlation analysis revealed that the family's co-evolution network was kept intact, which is one of the main advantages of full-length consensus design. The consensus enzymes displayed an "average" thermostability, that is, one that is higher than some but not all known representatives. We cautiously present practical rules for the design of consensus sequences, but warn that the measure of success depends on which natural enzyme is used as point of comparison. Biotechnol. Bioeng. 2013;110: 2563-2572. © 2013 Wiley Periodicals, Inc. Consensus engineering is supposed to increase a protein's stability by recruiting residues that occur most frequently at the respective positions in a set of homologous sequences. However, the authors have found that the most abundant residues do not generate the most stable construct but one that displays an average stability. Whether this strategy is successful thus depends on which of the parent sequences is used as a point of comparison. © 2013 Wiley Periodicals, Inc.

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