- Tom A. Ewing (3)
- Rienk A. Rienksma (1)
- Vítor A.P. Martins dos Santos (1)
- Ilona Alen van (1)
- Agnieszka B. Wegrzyn (1)
- Catarina Banha (1)
- Arjan Barendregt (2)
- Lubbert Dijkhuizen (1)
- Caroline E. Paul (2)
- Robert Geize van der (1)
- Gudrun Gygli (1)
- Eric Gädke (1)
- Adrie H. Westphal (3)
- Thomas Heine (1)
- Willem J.H. Berkel van (8)
- Willem J.H. Berkel Van (2)
- Albert J.R. Heck (2)
- Jasmin Kühn (1)
- Barbara M. Bakker (1)
- Mieke M.E. Huijbers (2)
- Joaquim Madeira (1)
- Maximino Manzanera (1)
- Andrea Mattevi (2)
- Sónia Mendes (1)
- Vanessa Miranda (1)
- Stefania Montersino (1)
- Aster Noord Van (1)
- Lígia O. Martins (1)
- Roberto Orru (1)
- Ronald P. Vries de (1)
- Evelien Poele te (1)
- Diana Santos (1)
- Anika Scholtissek (2)
- Silvia Segarra (1)
- Sarah Stolle (1)
- Maria Suarez-Diez (1)
- Dirk Tischler (2)
- Marta Tortajada (1)
- M.R. Ventura (1)
- Marco W. Fraaije (1)
- Jenny W. Wu (1)
- Ralf Zuhse (1)
Cofactors revisited – Predicting the impact of flavoprotein-related diseases on a genome scale
Wegrzyn, Agnieszka B. ; Stolle, Sarah ; Rienksma, Rienk A. ; Martins dos Santos, Vítor A.P. ; Bakker, Barbara M. ; Suarez-Diez, Maria - \ 2019
Biochimica et Biophysica Acta. Molecular Basis of Disease 1865 (2019)2. - ISSN 0925-4439 - p. 360 - 370.
Constraint-based modelling - FAD - Flavoprotein - FMN - Human genome-scale reconstruction - Inborn errors of metabolism
Flavin adenine dinucleotide (FAD) and its precursor flavin mononucleotide (FMN) are redox cofactors that are required for the activity of more than hundred human enzymes. Mutations in the genes encoding these proteins cause severe phenotypes, including a lack of energy supply and accumulation of toxic intermediates. Ideally, patients should be diagnosed before they show symptoms so that treatment and/or preventive care can start immediately. This can be achieved by standardized newborn screening tests. However, many of the flavin-related diseases lack appropriate biomarker profiles. Genome-scale metabolic models can aid in biomarker research by predicting altered profiles of potential biomarkers. Unfortunately, current models, including the most recent human metabolic reconstructions Recon and HMR, typically treat enzyme-bound flavins incorrectly as free metabolites. This in turn leads to artificial degrees of freedom in pathways that are strictly coupled. Here, we present a reconstruction of human metabolism with a curated and extended flavoproteome. To illustrate the functional consequences, we show that simulations with the curated model – unlike simulations with earlier Recon versions - correctly predict the metabolic impact of multiple-acyl-CoA-dehydrogenase deficiency as well as of systemic flavin-depletion. Moreover, simulations with the new model allowed us to identify a larger number of biomarkers in flavoproteome-related diseases, without loss of accuracy. We conclude that adequate inclusion of cofactors in constraint-based modelling contributes to higher precision in computational predictions.
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.
Multigram Scale Enzymatic Synthesis of (R)-1-(4′-Hydroxyphenyl)ethanol Using Vanillyl Alcohol Oxidase
Ewing, Tom A. ; Kühn, Jasmin ; Segarra, Silvia ; Tortajada, Marta ; Zuhse, Ralf ; Berkel, Willem J.H. van - \ 2018
Advanced Synthesis and Catalysis 360 (2018)12. - ISSN 1615-4150 - p. 2370 - 2376.
Alcohols - Alkylphenols - Enantioselectivity - Flavoprotein - Hydroxylation - Oxidoreductases
The enantioselective oxyfunctionalisation of C−H bonds is a highly interesting reaction, as it provides access to chiral alcohols that are important pharmaceutical building blocks. However, it is hard to achieve using traditional methods. One way in which it can be achieved is through the action of oxidative enzymes. Although many reports of the oxyfunctionalisation capabilities of enzymes at an analytical scale have been published, reports on the use of enzymes to achieve oxyfunctionalisation on a synthetically relevant scale are fewer. Here, we describe the scale-up of the conversion of 4-ethylphenol to (R)-1-(4′-hydroxyphenyl)ethanol using the flavin-dependent enzyme vanillyl alcohol oxidase. The process was optimised by testing different reaction media and substrate and enzyme concentrations and by performing it under an oxygen atmosphere. Under optimised reaction conditions, 4.10 g (R)-1-(4′-hydroxyphenyl)ethanol at 97% ee was obtained from 10 g 4-ethylphenol (isolated yield 36%). These results highlight some of the challenges that can be encountered during scale-up of an enzymatic oxyfunctionalisation process to a synthetically relevant scale and will be of use for the development of enzymatic processes for the synthesis of industrially relevant compounds. (Figure presented.).
On the origin of vanillyl alcohol oxidases
Gygli, Gudrun ; Vries, Ronald P. de; Berkel, Willem J.H. van - \ 2018
Fungal Genetics and Biology 116 (2018). - ISSN 1087-1845 - p. 24 - 32.
Dehydrogenase - Flavoprotein - Fungus - Phylogeny - Sequence motif
Vanillyl alcohol oxidase (VAO) is a fungal flavoenzyme that converts a wide range of para-substituted phenols. The products of these conversions, e.g. vanillin, coniferyl alcohol and chiral aryl alcohols, are of interest for several industries. VAO is the only known fungal member of the 4-phenol oxidising (4PO) subgroup of the VAO/PCMH flavoprotein family. While the enzyme has been biochemically characterised in great detail, little is known about its physiological role and distribution in fungi. We have identified and analysed novel, fungal candidate VAOs and found them to be mostly present in Pezizomycotina and Agaricomycotina. The VAOs group into three clades, of which two clades do not have any characterised member. Interestingly, bacterial relatives of VAO do not form a single outgroup, but rather split up into two separate clades. We have analysed the distribution of candidate VAOs in fungi, as well as their genomic environment. VAOs are present in low frequency in species of varying degrees of relatedness and in regions of low synteny. These findings suggest that fungal VAOs may have originated from bacterial ancestors, obtained by fungi through horizontal gene transfer. Because the overall conservation of fungal VAOs varies between 60 and 30% sequence identity, we argue for a more reliable functional prediction using critical amino acid residues. We have defined a sequence motif P-x-x-x-x-S-x-G-[RK]-N-x-G-Y-G-[GS] that specifically recognizes 4PO enzymes of the VAO/PCMH family, as well as additional motifs that can help to further narrow down putative functions. We also provide an overview of fingerprint residues that are specific to VAOs.
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.
A xylenol orange-based screening assay for the substrate specificity of flavin-dependent para-phenol oxidases
Ewing, Tom A. ; Noord, Aster Van; Paul, Caroline E. ; Berkel, Willem J.H. Van - \ 2018
Molecules 23 (2018)1. - ISSN 1420-3049
Enzyme kinetics - Flavoprotein - Oxidase - Screening assay - Substrate specificity
Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases.
N-terminus determines activity and specificity of styrene monooxygenase reductases
Heine, Thomas ; Scholtissek, Anika ; Westphal, Adrie H. ; Berkel, Willem J.H. van; Tischler, Dirk - \ 2017
Biochimica et Biophysica Acta. Proteins & Proteomics 1865 (2017)12. - ISSN 1570-9639 - p. 1770 - 1780.
Flavoprotein - Fluorescence spectroscopy - Fusion protein - Protein-ligand interaction - Rhodococcus opacus
Styrene monooxygenases (SMOs) are two-enzyme systems that catalyze the enantioselective epoxidation of styrene to (S)-styrene oxide. The FADH2 co-substrate of the epoxidase component (StyA) is supplied by an NADH-dependent flavin reductase (StyB). The genome of Rhodococcus opacus 1CP encodes two SMO systems. One system, which we define as E1-type, displays homology to the SMO from Pseudomonas taiwanensis VLB120. The other system, originally reported as a fused system (RoStyA2B), is defined as E2-type. Here we found that E1-type RoStyB is inhibited by FMN, while RoStyA2B is known to be active with FMN. To rationalize the observed specificity of RoStyB for FAD, we generated an artificial reductase, designated as RoStyBart, in which the first 22 amino acid residues of RoStyB were joined to the reductase part of RoStyA2B, while the oxygenase part (A2) was removed. RoStyBart mainly purified as apo-protein and mimicked RoStyB in being inhibited by FMN. Pre-incubation with FAD yielded a turnover number at 30 °C of 133.9 ± 3.5 s− 1, one of the highest rates observed for StyB reductases. RoStyBart holo-enzyme switches to a ping-pong mechanism and fluorescence analysis indicated for unproductive binding of FMN to the second (co-substrate) binding site. In summary, it is shown for the first time that optimization of the N-termini of StyB reductases allows the evolution of their activity and specificity.
The VAO/PCMH flavoprotein family
Ewing, Tom A. ; Fraaije, Marco W. ; Mattevi, Andrea ; Berkel, Willem J.H. van - \ 2017
Archives of Biochemistry and Biophysics 632 (2017). - ISSN 0003-9861 - p. 104 - 117.
Enzyme mechanism - Flavoprotein - Oxidoreductase - Phylogeny - Protein family
The VAO/PCMH flavoprotein family consists of structurally homologous flavin-dependent enzymes that catalyze a wide range of chemical reactions. Family members share an architecture consisting of a conserved FAD-binding domain and a more variable substrate-binding domain, which enables varying interactions with a range of substrates while maintaining the same cofactor-binding fold. Here, we provide an overview of the current state of our knowledge on the members of the VAO/PCMH family. Based on a phylogenetic analysis, we divide the family into 11 subgroups. We discuss the properties of these subgroups, focusing on recent developments in terms of the discovery of new family members and mechanistic advances. We also highlight open questions that will provide challenges for future research.
3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1 contains a phosphatidylinositol cofactor
Montersino, Stefania ; Poele, Evelien te; Orru, Roberto ; Westphal, Adrie H. ; Barendregt, Arjan ; Heck, Albert J.R. ; Geize, Robert van der; Dijkhuizen, Lubbert ; Mattevi, Andrea ; Berkel, Willem J.H. van - \ 2017
Frontiers in Microbiology 8 (2017). - ISSN 1664-302X - 11 p.
Expression strain - Flavoprotein - Monooxygenase - Phospholipid - Rhodococcus
3-Hydroxybenzoate 6-hydroxylase (3HB6H, EC 18.104.22.168) is a FAD-dependent monooxygenase involved in the catabolism of aromatic compounds in soil microorganisms. 3HB6H is unique among flavoprotein hydroxylases in that it harbors a phospholipid ligand. The purified protein obtained from expressing the gene encoding 3HB6H from Rhodococcus jostii RHA1 in the host Escherichia coli contains a mixture of phosphatidylglycerol and phosphatidylethanolamine, which are the major constituents of E. coli's cytoplasmic membrane. Here, we purified 3HB6H (RjHB6H) produced in the host R. jostii RHA#2 by employing a newly developed actinomycete expression system. Biochemical and biophysical analysis revealed that Rj3HB6H possesses similar catalytic and structural features as 3HB6H, but now contains phosphatidylinositol, which is a specific constituent of actinomycete membranes. Native mass spectrometry suggests that the lipid cofactor stabilizes monomer-monomer contact. Lipid analysis of 3HB6H from Pseudomonas alcaligenes NCIMB 9867 (Pa3HB6H) produced in E. coli supports the conclusion that 3HB6H enzymes have an intrinsic ability to bind phospholipids with different specificity, reflecting the membrane composition of their bacterial host.
Characterization of a bacterial pyranose 2-oxidase from Arthrobacter siccitolerans
Mendes, Sónia ; Banha, Catarina ; Madeira, Joaquim ; Santos, Diana ; Miranda, Vanessa ; Manzanera, Maximino ; Ventura, M.R. ; Berkel, Willem J.H. van; Martins, Lígia O. - \ 2016
Journal of Molecular Catalysis. B, Enzymatic 133 (2016)suppl. 1. - ISSN 1381-1177 - p. S34 - S43.
Carbohydrate chemistry - Flavoprotein - Glucose-methanol-choline family of oxidoreductases - Hydrogen peroxide forming enzymes
In this study we provide the first biochemical characterization of a bacterial pyranose 2-oxidase (AsP2Ox) from Arthrobacter siccitolerans. The enzyme catalyzes the oxidation of several aldopyranoses at the C-2 position, coupling it to the reduction of dioxygen to hydrogen peroxide. Pyranose 2-oxidases belong to the glucose-methanol-choline oxidoreductase family. A structural model based on the known X-ray structure of P2Ox from Phanerochaete chrysosporium supports that AsP2Ox shares structural features with well-characterized fungal P2Oxs. The gene coding for AsP2Ox was cloned and heterologously expressed in Escherichia coli. The purified recombinant enzyme is a 64-kDa monomer containing a non-covalently bound flavin adenine dinucleotide (FAD) cofactor, distinct features as compared with fungal counterparts that are ∼ 270kDa homotetramers with covalent-linked FAD. AsP2Ox exhibits a redox potential of -50mV, an optimum temperature of 37C and an optimum pH at 6.5. AsP2Ox oxidizes d-glucose at the highest efficiency, using additionally d-galactose, d-xylose, l-arabinose and d-ribose as electron donors, coupling their oxidation to the reduction of both dioxygen and 1,4-benzoquinone. AsP2Ox shows a relatively low thermal stability with a melting temperature (T m) of 43C and a half-life (t1/2) at 40C of 25min. This work expands the repertoire of bacterial oxidoreductases with importance in biotechnological and diagnostic applications.
A more polar N-terminal helix releases MBP-tagged Thermus thermophilus proline dehydrogenase from tetramer-polymer self-association
Huijbers, Mieke M.E. ; Berkel, Willem J.H. van - \ 2016
Journal of Molecular Catalysis. B, Enzymatic 134 (2016). - ISSN 1381-1177 - p. 340 - 346.
Flavoprotein - Molecular self-association - Proline dehydrogenase - Protein oligomerization - Thermus thermophilus
Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme involved in the biosynthesis of . l-glutamate. ProDH is of interest for biocatalysis because the protein might be applied in multi-enzyme reactions for the synthesis of structurally complex molecules. We recently demonstrated that the thermotolerant ProDH from . Thermus thermophilus (TtProDH) is overproduced in . Escherichia coli when using maltose-binding protein (MBP) as a solubility tag. However, MBP-TtProDH and MBP-clipped TtProDH are prone to aggregation through non-native self-association. Here we provide evidence that the hydrophobic N-terminal helix of TtProDH is responsible for the self-association process. The more polar MBP-tagged F10E/L12E variant exclusively forms tetramers and exhibits excellent catalytic features over a wide range of temperatures. Understanding the hydrodynamic and catalytic properties of thermostable enzymes is important for the development of industrial biocatalysts as well as for pharmaceutical applications.