Wageningen, Netherlands
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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).


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.


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.


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.


Nobili A.,University of Greifswald | Tao Y.,University of Greifswald | Tao Y.,Beijing University of Chemical Technology | Pavlidis I.V.,University of Greifswald | And 4 more authors.
ChemBioChem | Year: 2015

In order to improve the efficiency of directed evolution experiments, in silico multiple-substrate clustering was combined with an analysis of the variability of natural enzymes within a protein superfamily. This was applied to a Pseudomonas fluorescens esterase (PFE I) targeting the enantioselective hydrolysis of 3-phenylbutyric acid esters. Data reported in the literature for nine substrates were used for the clustering meta-analysis of the docking conformations in wild-type PFE I, and this highlighted a tryptophan residue (W28) as an interesting target. Exploration of the most frequently, naturally occurring amino acids at this position suggested that the reduced flexibility observed in the case of the W28F variant leads to enhancement of the enantioselectivity. This mutant was subsequently combined with mutations identified in a library based on analysis of a correlated mutation network. By interrogation of <80 variants a mutant with 15-fold improved enantioselectivity was found. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Genz M.,University of Greifswald | Melse O.,University of Greifswald | Schmidt S.,University of Greifswald | Vickers C.,University of Greifswald | And 4 more authors.
ChemCatChem | Year: 2016

Chiral amines are important building blocks, especially for the pharmaceutical industry. Although amine transaminases (ATAs) are versatile enzymes to synthesize chiral amines, the wildtype enzymes do not accept ketones with two large substituents next to the carbonyl functionality. Using bioinformatic tools to design a seven-site mutant library followed by high-throughput screening, we were able to identify variants of the enzyme from Vibrio fluvialis (VF-ATA) with a widened binding pocket, as exemplified for a range of ketones. Three variants allowed the asymmetric synthesis of 2,2-dimethyl-1-phenylpropan-1-amine—not accessible by any wildtype ATA described so far. The best variant containing four mutations (L56V, W57C, F85V, V153A) gave 100 % conversion of the ketone to yield the amine with an enantiomeric excess value >99 %, notably with preference for the (R)-enantiomer. In silico modeling enabled the reconstruction of the substrate binding mode to the newly evolved pocket and, hence, allowed explanation of the experimental results. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


Steffen-Munsberg F.,University of Greifswald | Steffen-Munsberg F.,Albanova University Center | Vickers C.,University of Greifswald | Kohls H.,University of Greifswald | And 9 more authors.
Biotechnology Advances | Year: 2015

In this review we analyse structure/sequence-function relationships for the superfamily of PLP-dependent enzymes with special emphasis on class III transaminases. Amine transaminases are highly important for applications in biocatalysis in the synthesis of chiral amines. In addition, other enzyme activities such as racemases or decarboxylases are also discussed. The substrate scope and the ability to accept chemically different types of substrates are shown to be reflected in conserved patterns of amino acids around the active site. These findings are condensed in a sequence-function matrix, which facilitates annotation and identification of biocatalytically relevant enzymes and protein engineering thereof. © 2015 Elsevier Inc.


PubMed | University of Greifswald and Bio Prodict
Type: | Journal: Applied microbiology and biotechnology | Year: 2016

Pyridoxal-5-phosphate (PLP)-dependent enzymes are ubiquitous in nature and catalyze a variety of important metabolic reactions. The fold-type III PLP-dependent enzyme family is primarily comprised of decarboxylases and alanine racemases. In the development of a multiple structural alignment database (3DM) for the enzyme family, a large subset of 5666 uncharacterized proteins with high structural, but low sequence similarity to alanine racemase and decarboxylases was found. Compared to these two classes of enzymes, the protein sequences being the object of this study completely lack the C-terminal domain, which has been reported important for the formation of the dimer interface in other fold-type III enzymes. The 5666 sequences cluster around four protein templates, which also share little sequence identity to each other. In this work, these four template proteins were solubly expressed in Escherichia coli, purified, and their substrate profiles were evaluated by HPLC analysis for racemase activity using a broader range of amino acids. They were found active only against alanine or serine, where they exhibited Michaelis constants within the range of typical bacterial alanine racemases, but with significantly lower turnover numbers. As the already described racemases were proposed to be active and appeared to be monomers as judged from their crystal structures, we also investigated this aspect for the four new enzymes. Here, size exclusion chromatography indicated the presence of oligomeric states of the enzymes and a native-PAGE in-gel assay showed that the racemase activity was present only in an oligomeric state but not as monomer. This suggests the likelihood of a different behavior of these enzymes in solution compared to the one observed in crystalline form.


PubMed | University of Greifswald and Bio Prodict
Type: Journal Article | Journal: International journal of molecular sciences | Year: 2015

To alter the amine donor/acceptor spectrum of an (S)-selective amine transaminase (ATA), a library based on the Vibrio fluvialis ATA targeting four residues close to the active site (L56, W57, R415 and L417) was created. A 3DM-derived alignment comprising fold class I pyridoxal-5-phosphate (PLP)-dependent enzymes allowed identification of positions, which were assumed to determine substrate specificity. These positions were targeted for mutagenesis with a focused alphabet of hydrophobic amino acids to convert an amine:-keto acid transferase into an amine:aldehyde transferase. Screening of 1200 variants revealed three hits, which showed a shifted amine donor/acceptor spectrum towards aliphatic aldehydes (mainly pentanal), as well as an altered pH profile. Interestingly, all three hits, although found independently, contained the same mutation R415L and additional W57F and L417V substitutions.


PubMed | University of Greifswald and Bio Prodict
Type: | Journal: Chembiochem : a European journal of chemical biology | Year: 2016

Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. TypeI BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO

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