Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    We will mail you new results for this query: keywords==Protein engineering
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Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcusfuriosus Phosphoglucose Isomerase
Subramanian, Kalyanasundaram ; Mitusińska, Karolina ; Raedts, John ; Almourfi, Feras ; Joosten, Henk Jan ; Hendriks, Sjon ; Sedelnikova, Svetlana E. ; Kengen, Servé W.M. ; Hagen, Wilfred R. ; Góra, Artur ; Martins Dos Santos, Vitor A.P. ; Baker, Patrick J. ; Oost, John van der; Schaap, Peter J. - \ 2019
Biomolecules 9 (2019)6. - ISSN 2218-273X
Comulator - cupin phosphoglucose isomerase - Protein engineering - Pyrococcus furiosus - solvent access

The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.

Catalytic performance of a class III Old yellow enzyme and its cysteine variants
Scholtissek, Anika ; Gädke, Eric ; Paul, Caroline E. ; Westphal, Adrie H. ; Berkel, Willem J.H. van; Tischler, Dirk - \ 2018
Frontiers in Microbiology 9 (2018)OCT. - ISSN 1664-302X
Actinobacteria - Biocatalysis - Cysteine modification - Ene reductase - Flavoprotein - Inactivation - Protein engineering - Rhodococcus opacus 1CP

Class III old yellow enzymes (OYEs) contain a conserved cysteine in their active sites. To address the role of this cysteine in OYE-mediated asymmetric synthesis, we have studied the biocatalytic properties of OYERo2a from Rhodococcus opacus 1CP (WT) as well as its engineered variants C25A, C25S and C25G. OYERo2a in its redox resting state (oxidized form) is irreversibly inactivated by N-methylmaleimide. As anticipated, inactivation does not occur with the Cys variants. Steady-state kinetics with this maleimide substrate revealed that C25S and C25G doubled the turnover frequency (kcat) while showing increased KM values compared to WT, and that C25A performed more similar to WT. Applying the substrate 2-cyclohexen-1-one, the Cys variants were less active and less efficient than WT. OYERo2a and its Cys variants showed different activities with NADPH, the natural reductant. The variants did bind NADPH less well but kcat was significantly increased. The most efficient variant was C25G. Replacement of NADPH with the cost-effective synthetic cofactor 1-benzyl-1,4-dihydronicotinamide (BNAH) drastically changed the catalytic behavior. Again C25G was most active and showed a similar efficiency as WT. Biocatalysis experiments showed that OYERo2a, C25S, and C25G converted N-phenyl-2-methylmaleimide equally well (81-84%) with an enantiomeric excess (ee) of more than 99% for the R-product. With cyclic ketones, the highest conversion (89%) and ee (>99%) was observed for the reaction of WT with R-carvone. A remarkable poor conversion of cyclic ketones occurred with C25G. In summary, we established that the generation of a cysteine-free enzyme and cofactor optimization allows the development of more robust class III OYEs.

In vivo selection of sfGFP variants with improved and reliable functionality in industrially important thermophilic bacteria
Frenzel, Elrike ; Legebeke, Jelmer ; Stralen, Atze Van; Kranenburg, Richard Van; Kuipers, Oscar P. - \ 2018
Biotechnology for Biofuels 11 (2018)1. - ISSN 1754-6834
Biotechnology - Cyan - FACS - Geobacillus sp. - GFP - In vivo application - Parageobacillus sp. - Protein engineering - sfGFP - Thermophilic bacteria - Thermostability - Yellow
Background: Fluorescent reporter proteins (FP) have become an indispensable tool for the optimization of microbial cell factories and in synthetic biology per se. The applicability of the currently available FPs is, however, constrained by species-dependent performance and misfolding at elevated temperatures. To obtain functional reporters for thermophilic, biotechnologically important bacteria such as Parageobacillus thermoglucosidasius, an in vivo screening approach based on a mutational library of superfolder GFP was applied. Results: Flow cytometry-based benchmarking of a set of GFPs, sfGFPs and species-specific codon-optimized variants revealed that none of the proteins was satisfyingly detectable in P. thermoglucosidasius at its optimal growth temperature of 60 °C. An undirected mutagenesis approach coupled to fluorescence-activated cell sorting allowed the isolation of sfGFP variants that were extremely well expressed in the chassis background at 60 °C. Notably, a few nucleotide substitutions, including silent mutations, significantly improved the functionality and brightness. The best mutant sfGFP(N39D/A179A) showed an 885-fold enhanced mean fluorescence intensity (MFI) at 60 °C and is the most reliable reporter protein with respect to cell-to-cell variation and signal intensity reported so far. The in vitro spectral and thermostability properties were unaltered as compared to the parental sfGFP protein, strongly indicating that the combination of the amino acid exchange and an altered translation or folding speed, or protection from degradation, contribute to the strongly improved in vivo performance. Furthermore, sfGFP(N39D/A179A) and the newly developed cyan and yellow derivatives were successfully used for labeling several industrially relevant thermophilic bacilli, thus proving their broad applicability. Conclusions: This study illustrates the power of in vivo isolation of thermostable proteins to obtain reporters for highly efficient fluorescence labeling. Successful expression in a variety of thermophilic bacteria proved that the novel FPs are highly suitable for imaging and flow cytometry-based studies. This enables a reliable cell tracking and single-cell-based real-time monitoring of biological processes that are of industrial and biotechnological interest.
Functional impact of the n-terminal arm of proline dehydrogenase from thermus thermophilus
Huijbers, Mieke M.E. ; Alen, Ilona van; Wu, Jenny W. ; Barendregt, Arjan ; Heck, Albert J.R. ; Berkel, Willem J.H. Van - \ 2018
Molecules 23 (2018)1. - ISSN 1420-3049
Flavoprotein - Proline dehydrogenase - Protein engineering - Protein oligomerization - Solubility tag - Suicide inhibition - TIM-barrel
Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme that catalyzes the oxidation of proline to ∆1-pyrroline-5-carboxylate. Thermus thermophilus ProDH (TtProDH) contains in addition to its flavin-binding domain an N-terminal arm, consisting of helices αA, αB, and αC. Here, we report the biochemical properties of the helical arm truncated TtProDH variants ∆A, ∆AB, and ∆ABC, produced with maltose-binding protein as solubility tag. All three truncated variants show similar spectral properties as TtProDH, indicative of a conserved flavin-binding pocket. ∆A and ∆AB are highly active tetramers that rapidly react with the suicide inhibitor N-propargylglycine. Removal of the entire N-terminal arm (∆ABC) results in barely active dimers that are incapable of forming a flavin adduct with N-propargylglycine. Characterization of V32D, Y35F, and V36D variants of ∆AB established that a hydrophobic patch between helix αC and helix α8 is critical for TtProDH catalysis and tetramer stabilization.
Supramolecular Virus-Like Nanorods by Coassembly of a Triblock Polypeptide and Reversible Coordination Polymers
Hernandez-Garcia, Armando ; Velders, Aldrik H. ; Cohen Stuart, Martien ; Vries, Renko de; Lent, Jan van; Wang, Junyou - \ 2017
Chemistry-A European Journal 23 (2017)2. - ISSN 0947-6539 - p. 239 - 243.
Coordination polymers - Hybrid nanomaterials - Polypeptides - Protein engineering - Self-assembly

We investigate a new case of a self-assembly-stimulated self-assembly in which a triblock polypeptide is combined with a anionic coordination polymer of a dipicolinic acid bis-ligand, and d- or f- block metal ions like ZnII or EuIII. The polypeptide not only has a silk-like domain that can fold and stack, but also a C-terminal cationic sequence by which it can interact with the supramolecular (coordination) polyanion. In the presence of all three ingredients (polypeptide, bis-ligand, and metal ions), we observe the initiation and slow growth of well-defined metal-containing nanorods of up to 150nm in length, proving that self-assembly of the polypeptide is triggered by the self-assembly of the coordination polyelectrolyte and vice versa. The particles, which have a striking resemblance to rod-like viruses, are stable up to 1.2m NaCl, and can be made fluorescent when lanthanides like EuIII are used, showing the potential to exploit functional properties and applications of virus-like supramolecular structures.

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