Hein Wijma - Academia.edu (original) (raw)

Papers by Hein Wijma

Research paper thumbnail of Computational Library Design for Increasing Haloalkane Dehalogenase Stability

ChemBioChem, 2014

We explored the use of a computational design framework for the stabilization of the haloalkane d... more We explored the use of a computational design framework for the stabilization of the haloalkane dehalogenase LinB. Energy calculations, disulfide bond design, molecular dynamics simulations, and rational inspection of mutant structures predicted many stabilizing mutations. Screening of these in small mutant libraries led to the discovery of seventeen point mutations and one disulfide bond that enhanced thermostability. Mutations located in or contacting flexible regions of the protein had a larger stabilizing effect than mutations outside such regions. The combined introduction of twelve stabilizing mutations resulted in a LinB mutant with a 23 °C increase in apparent melting temperature (Tm,app , 72.5 °C) and an over 200-fold longer half-life at 60 °C. The most stable LinB variants also displayed increased compatibility with co-solvents, thus allowing substrate conversion and kinetic resolution at much higher concentrations than with the wild-type enzyme.

Research paper thumbnail of A Rearranging Ligand Enables Allosteric Control of Catalytic Activity in Copper-containing Nitrite Reductase

Journal of Molecular Biology, 2006

In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of t... more In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of the type-1 site was replaced (M150G) to make the copper ion accessible to external ligands that might affect the enzyme's catalytic activity. The type-1 site optical spectrum of M150G (A 460 /A 600 Z 0.71) differs significantly from that of the native nitrite reductase (A 460 / A 600 Z1.3). The midpoint potential of the type-1 site of nitrite reductase M150G (E M Z312(G5) mV versus hydrogen) is higher than that of the native enzyme (E M Z213(G5) mV). M150G has a lower catalytic activity (k cat Z 133(G6) s K1 ) than the wild-type nitrite reductase (k cat Z416(G10) s K1 ). The binding of external ligands to M150G restores spectral properties, midpoint potential (E M !225 mV), and catalytic activity (k cat Z374(G28) s K1 ). Also the M150H (A 460 /A 600 Z7.7, E M Z104(G5) mV, k cat Z0.099(G0.006) s K1 ) and M150T (A 460 /A 600 Z0.085, E M Z340(G5) mV, k cat Z126(G2) s K1 ) variants were characterized. Crystal structures show that the ligands act as allosteric effectors by displacing Met62, which moves to bind to the Cu in the position emptied by the M150G mutation. The reconstituted type-1 site has an otherwise unaltered geometry. The observation that removal of an endogenous ligand can introduce allosteric control in a redox enzyme suggests potential for structural and functional flexibility of coppercontaining redox sites.

Research paper thumbnail of Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase

Biochimica Et Biophysica Acta Proteins and Proteomics, Aug 1, 2005

Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types ... more Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type 1 is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes ...

Research paper thumbnail of Computationally efficient and accurate enantioselectivity modeling by clusters of molecular dynamics simulations

Journal of chemical information and modeling, Jan 28, 2014

Computational approaches could decrease the need for the laborious high-throughput experimental s... more Computational approaches could decrease the need for the laborious high-throughput experimental screening that is often required to improve enzymes by mutagenesis. Here, we report that using multiple short molecular dynamics (MD) simulations makes it possible to accurately model enantioselectivity for large numbers of enzyme-substrate combinations at low computational costs. We chose four different haloalkane dehalogenases as model systems because of the availability of a large set of experimental data on the enantioselective conversion of 45 different substrates. To model the enantioselectivity, we quantified the frequency of occurrence of catalytically productive conformations (near attack conformations) for pairs of enantiomers during MD simulations. We found that the angle of nucleophilic attack that leads to carbon-halogen bond cleavage was a critical variable that limited the occurrence of productive conformations; enantiomers for which this angle reached values close to 180° ...

Research paper thumbnail of Enantioselective Enzymes by Computational Design and In Silico Screening

Angewandte Chemie (International ed. in English), Jan 4, 2015

Computational enzyme design holds great promise for providing new biocatalysts for synthetic chem... more Computational enzyme design holds great promise for providing new biocatalysts for synthetic chemistry. A strategy to design small mutant libraries of complementary enantioselective epoxide hydrolase variants for the production of highly enantioenriched (S,S)-diols and (R,R)-diols is developed. Key features of this strategy (CASCO, catalytic selectivity by computational design) are the design of mutations that favor binding of the substrate in a predefined orientation, the introduction of steric hindrance to prevent unwanted substrate binding modes, and ranking of designs by high-throughput molecular dynamics simulations. Using this strategy we obtained highly stereoselective mutants of limonene epoxide hydrolase after experimental screening of only 37 variants. The results indicate that computational methods can replace a substantial amount of laboratory work when developing enantioselective enzymes.

Research paper thumbnail of Stabilization of cyclohexanone monooxygenase by a computationally designed disulfide bond spanning only one residue

FEBS Open Bio, 2014

Enzyme stability is an important parameter in biocatalytic applications, and there is a strong ne... more Enzyme stability is an important parameter in biocatalytic applications, and there is a strong need for efficient methods to generate robust enzymes. We investigated whether stabilizing disulfide bonds can be computationally designed based on a model structure. In our approach, unlike in previous disulfide engineering studies, short bonds spanning only a few residues were included. We used cyclohexanone monooxygenase (CHMO), a Baeyer-Villiger monooxygenase (BVMO) from Acinetobacter sp. NCIMB9871 as the target enzyme. This enzyme has been the prototype BVMO for many biocatalytic studies even though it is notoriously labile. After creating a small library of mutant enzymes with introduced cysteine pairs and subsequent screening for improved thermostability, three stabilizing disulfide bonds were identified. The introduced disulfide bonds are all within 12 Å of each other, suggesting this particular region is critical for unfolding. This study shows that stabilizing disulfide bonds do not have to span many residues, as the most stabilizing disulfide bond, L323C-A325C, spans only one residue while it stabilizes the enzyme, as shown by a 6°C increase in its apparent melting temperature.

Research paper thumbnail of Computational design gains momentum in enzyme catalysis engineering

FEBS Journal, 2013

Computational protein design is becoming a powerful tool for tailoring enzymes for specific biote... more Computational protein design is becoming a powerful tool for tailoring enzymes for specific biotechnological applications. When applied to existing enzymes, computational re-design makes it possible to obtain orders of magnitude improvement in catalytic activity towards a new target substrate. Computational methods also allow the design of completely new active sites that catalyze reactions that are not known to occur in biological systems. If initial designs display modest catalytic activity, which is often the case, this may be improved by iterative cycles of computational design or by follow-up engineering through directed evolution. Compared to established protein engineering methods such as directed evolution and structure-based mutagenesis, computational design allows for much larger jumps in sequence space; for example, by introducing more than a dozen mutations in a single step or by introducing loops that provide new functional interactions. Recent advances in the computational design toolbox, which include new backbone re-design methods and the use of molecular dynamics simulations to better predict the catalytic activity of designed variants, will further enhance the use of computational tools in enzyme engineering.

Research paper thumbnail of Computationally designed libraries for rapid enzyme stabilization

Protein Engineering Design and Selection, 2014

The ability to engineer enzymes and other proteins to any desired stability would have wide-rangi... more The ability to engineer enzymes and other proteins to any desired stability would have wide-ranging applications.

Research paper thumbnail of Protein Film Voltammetry of Copper-Containing Nitrite Reductase Reveals Reversible Inactivation

Journal of the American Chemical Society, 2007

The Cu-containing nitrite reductase from Alcaligenes faecalis S-6 catalyzes the one-electron redu... more The Cu-containing nitrite reductase from Alcaligenes faecalis S-6 catalyzes the one-electron reduction of nitrite to nitric oxide (NO). Electrons enter the enzyme at the so-called type-1 Cu site and are then transferred internally to the catalytic type-2 Cu site. Protein film voltammetry experiments were carried out to obtain detailed information about the catalytic cycle. The homotrimeric structure of the enzyme is reflected in a distribution of the heterogeneous electron-transfer rates around three main values. Otherwise, the properties and the mode of operation of the enzyme when it is adsorbed as a film on a pyrolytic graphite electrode are essentially unchanged compared to those of the free enzyme in solution. It was established that the reduced type-2 site exists in either an active or an inactive conformation with an interconversion rate of approximately 0.1 s(-1). The random sequential mechanism comprises two routes, one in which the type-2 site is reduced first and subsequently binds nitrite, which is then converted into NO, and another in which the oxidized type-2 site binds nitrite and then accepts an electron to produce NO. At high nitrite concentration, the second route prevails and internal electron transfer is rate-limiting. The midpoint potentials of both sites could be established under catalytic conditions. Binding of nitrite to the type-2 site does not affect the midpoint potential of the type-1 site, thereby excluding cooperativity between the two sites.

Research paper thumbnail of Effect of the Methionine Ligand on the Reorganization Energy of the Type-1 Copper Site of Nitrite Reductase

Journal of the American Chemical Society, 2007

Copper-containing nitrite reductase harbors a type-1 and a type-2 Cu site. The former acts as the... more Copper-containing nitrite reductase harbors a type-1 and a type-2 Cu site. The former acts as the electron acceptor site of the enzyme, and the latter is the site of catalytic action. The effect of the methionine ligand on the reorganization energy of the type-1 site was explored by studying the electrontransfer kinetics between NiR (wild type (wt) and the variants Met150Gly and Met150Thr) with Fe(II)EDTA and Fe(II)HEDTA. The mutations increased the reorganization energy by 0.3 eV (30 kJ mol -1 ). A similar increase was found from pulse radiolysis experiments on the wt NIR and three variants (Met150Gly, Met150His, and Met150Thr). Binding of the nearby Met62 to the type-1 Cu site in Met150Gly (under influence of an allosteric effector) lowered the reorganization energy back to approximately the wt value. According to XRD data the structure of the reduced type-1 site in Met150Gly NiR in the presence of an allosteric effector is similar to that in the reduced wt NiR (solved to 1.85 Å), compatible with the similarity in reorganization energy.

Research paper thumbnail of A Rearranging Ligand Enables Allosteric Control of Catalytic Activity in Copper-containing Nitrite Reductase

Journal of Molecular Biology, 2006

In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of t... more In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of the type-1 site was replaced (M150G) to make the copper ion accessible to external ligands that might affect the enzyme's catalytic activity. The type-1 site optical spectrum of M150G (A(460)/A(600)=0.71) differs significantly from that of the native nitrite reductase (A(460)/A(600)=1.3). The midpoint potential of the type-1 site of nitrite reductase M150G (E(M)=312(+/-5)mV versus hydrogen) is higher than that of the native enzyme (E(M)=213(+/-5)mV). M150G has a lower catalytic activity (k(cat)=133(+/-6)s(-1)) than the wild-type nitrite reductase (k(cat)=416(+/-10)s(-1)). The binding of external ligands to M150G restores spectral properties, midpoint potential (E(M)<225mV), and catalytic activity (k(cat)=374(+/-28)s(-1)). Also the M150H (A(460)/A(600)=7.7, E(M)=104(+/-5)mV, k(cat)=0.099(+/-0.006)s(-1)) and M150T (A(460)/A(600)=0.085, E(M)=340(+/-5)mV, k(cat)=126(+/-2)s(-1)) variants were characterized. Crystal structures show that the ligands act as allosteric effectors by displacing Met62, which moves to bind to the Cu in the position emptied by the M150G mutation. The reconstituted type-1 site has an otherwise unaltered geometry. The observation that removal of an endogenous ligand can introduce allosteric control in a redox enzyme suggests potential for structural and functional flexibility of copper-containing redox sites.

Research paper thumbnail of A Random-sequential Mechanism for Nitrite Binding and Active Site Reduction in Copper-containing Nitrite Reductase

Journal of Biological Chemistry, 2006

The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 cop... more The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of pH and nitrite on the turnover rate in the presence of three different electron donors at saturating concentrations were measured. The activity of NiR was also measured electrochemically by exploiting direct electron transfer to the enzyme immobilized on a graphite rotating disk electrode. In all cases, the steady-state kinetics fitted excellently to a random-sequential mechanism in which electron transfer from the type-1 to the type-2 site is rate-limiting. At low [NO(-)(2)] reduction of the type-2 site precedes nitrite binding, at high [NO(-)(2)] the reverse occurs. Below pH 6.5, the catalytic activity diminished at higher nitrite concentrations, in agreement with electron transfer being slower to the nitrite-bound type-2 site than to the water-bound type-2 site. Above pH 6.5, substrate activation is observed, in agreement with electron transfer to the nitrite-bound type-2 site being faster than electron transfer to the hydroxyl-bound type-2 site. To study the effect of slower electron transfer between the type-1 and type-2 site, NiR M150T was used. It has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an approximately 50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism.

Research paper thumbnail of Aminoacyl-coenzyme A synthesis catalyzed by a CoA ligase from Penicillium chrysogenum

FEBS Letters, 2011

Keywords: Coenzyme A ligase Aminoacyl-coenzyme A b-Phenylalanine Penicillium chrysogenum a b s t ... more Keywords: Coenzyme A ligase Aminoacyl-coenzyme A b-Phenylalanine Penicillium chrysogenum a b s t r a c t Coenzyme A ligases play an important role in metabolism by catalyzing the activation of carboxylic acids. In this study we describe the synthesis of aminoacyl-coenzyme As (CoAs) catalyzed by a CoA ligase from Penicillium chrysogenum. The enzyme accepted medium-chain length fatty acids as the best substrates, but the proteinogenic amino acids L-phenylalanine and L-tyrosine, as well as the non-proteinogenic amino acids D-phenylalanine, D-tyrosine and (R)-and (S)-b-phenylalanine were also accepted. Of these amino acids, the highest activity was found for (R)-b-phenylalanine, forming (R)-b-phenylalanyl-CoA. Homology modeling suggested that alanine 312 is part of the active site cavity, and mutagenesis (A312G) yielded a variant that has an enhanced catalytic efficiency with b-phenylalanines and D-a-phenylalanine.

Research paper thumbnail of Thermal stability effects of removing the type-2 copper ligand His306 at the interface of nitrite reductase subunits

European Biophysics Journal, 2007

Nitrite reductase (NiR) is a highly stable trimeric protein, which denatures via an intermediate,... more Nitrite reductase (NiR) is a highly stable trimeric protein, which denatures via an intermediate, U-unfolded and F-final). To understand the role of interfacial residues on protein stability, a type-2 copper site ligand, His306, has been mutated to an alanine. The characterization of the native state of the mutated protein highlights that this mutation prevents copper ions from binding to the type-2 site and eliminates catalytic activity. No significant alteration of the geometry of the type-1 site is observed. Study of the thermal denaturation of this His306Ala NiR variant by differential scanning calorimetry shows an endothermic irreversible profile, with maximum heat absorption at T max % 85°C, i.e., 15°C lower than the corresponding value found for wild-type protein. The reduction of the protein thermal stability induced by the His306Ala replacement was also shown by optical spectroscopy. The denaturation pathway of the variant is compatible with the kinetic model N 3 ! k F 3 ; where the protein irreversibly passes from the native to the final state. No evidence of subunits' dissociation has been found within the unfolding process. The results show that the type-2 copper sites, situated at the interface of two monomers, significantly contribute to both the stability and the denaturation mechanism of NiR.

Research paper thumbnail of Structure- and sequence-analysis inspired engineering of proteins for enhanced thermostability

Current Opinion in Structural Biology, 2013

Protein engineering strategies for increasing stability can be improved by replacing random mutag... more Protein engineering strategies for increasing stability can be improved by replacing random mutagenesis and high-throughput screening by approaches that include bioinformatics and computational design. Mutations can be focused on regions in the structure that are most flexible and involved in the early steps of thermal unfolding. Sequence analysis can often predict the position and nature of stabilizing mutations, and may allow the reconstruction of thermostable ancestral sequences. Various computational tools make it possible to design stabilizing features, such as hydrophobic clusters and surface charges. Different methods for designing chimeric enzymes can also support the engineering of more stable proteins without the need of high-throughput screening.

Research paper thumbnail of Directed Evolution Strategies for Enantiocomplementary Haloalkane Dehalogenases: From Chemical Waste to Enantiopure Building Blocks

Research paper thumbnail of Bidirectional Catalysis by Copper-Containing Nitrite Reductase †

Biochemistry, 2004

The copper-containing nitrite reductase from Alcaligenes faecalis S-6 was found to catalyze the o... more The copper-containing nitrite reductase from Alcaligenes faecalis S-6 was found to catalyze the oxidation of nitric oxide to nitrite, the reverse of its physiological reaction. Thermodynamic and kinetic constants with the physiological electron donor pseudoazurin were determined for both directions of the catalyzed reaction in the pH range of 6-8. For this, nitric oxide was monitored by a Clark-type electrode, and the redox state of pseudoazurin was measured by optical spectroscopy. The equilibrium constant (K(eq)) depends on the reduction potentials of pseudoazurin and nitrite/nitric oxide, both of which vary with pH. Above pH 6.2 the formation of NiR substrates (nitrite and reduced pseudoazurin) is favored over the products (NO and oxidized pseudoazurin). At pH 8 the K(eq) amounts to 10(3). The results show that dissimilatory nitrite reductases catalyze an unfavorable reaction at physiological pH (pH = 7-8). Consequently, nitrous oxide production by copper-containing nitrite reductases is unlikely to occur in vivo with a native electron donor. With increasing pH, the rate and specificity constant of the forward reaction decrease and become lower than the rate of the reverse reaction. The opposite occurs for the rate of the reverse reaction; thus the catalytic bias for nitrite reduction decreases. At pH 6.0 the k(cat) for nitrite reduction was determined to be 1.5 x 10(3) s(-1), and at pH 8 the rate of the reverse reaction is 125 s(-1).

Research paper thumbnail of Reconstitution of the Type-1 Active Site of the H145G/A Variants of Nitrite Reductase by Ligand Insertion †

Biochemistry, 2003

Variants of the copper-containing nitrite reductase (NiR) of Alcaligenes faecalis S6 were constru... more Variants of the copper-containing nitrite reductase (NiR) of Alcaligenes faecalis S6 were constructed by site-directed mutagenesis, by which the C-terminal histidine ligand (His145) of the Cu in the type-1 site was replaced by an alanine or a glycine. The type-1 sites in the NiR variants as isolated, are in the reduced form, but can be oxidized in the presence of external ligands, like (substituted) imidazoles and chloride. The reduction potential of the type-1 site of NiR-H145A reconstituted with imidazole amounts to 505 mV vs NHE (20 degrees C, pH 7, 10 mM imidazole), while for the native type-1 site it amounts to 260 mV. XRD data on crystals of the reduced and oxidized NiR-H145A variant show that in the reduced type-1 site the metal is 3-coordinated, but in the oxidized form takes up a ligand from the solution. With the fourth (exogenous) ligand in place the type-1 site is able to accept electrons at about the same rate as the wt NiR, but it is unable to pass the electron onto the type-2 site, leading to loss of enzymatic activity. It is argued that the uptake of an electron by the mutated type-1 site is accompanied by a loss of the exogenous ligand and a concomitant rise of the redox potential. This rise effectively traps the electron in the type-1 site.

Research paper thumbnail of The Substrate-Bound Type 2 Copper Site of Nitrite Reductase:  The Nitrogen Hyperfine Coupling of Nitrite Revealed by Pulsed EPR †

Biochemistry, 2005

A pulsed electron paramagnetic resonance study has been performed on the type 2 copper site of ni... more A pulsed electron paramagnetic resonance study has been performed on the type 2 copper site of nitrite reductase (NiR) from Alcaligenes faecalis. The H145A mutant, in which histidine 145 is replaced by alanine, was studied by ESEEM and HYSCORE experiments at 9 GHz on frozen solutions. This mutant contains a reduced type 1 copper site which allowed a selective investigation of the type 2 site of H145A and of its nitrite-bound form H145A (NO2(-)). The experiments yielded hyperfine and quadrupole parameters of the remote nitrogens of two of the histidines in the type 2 copper site of the protein and revealed the changes of these values induced by substrate binding (14NO2(-) and 15NO2(-)). The HYSCORE experiments displayed a signal of 15NO2(-) bound to H145A, from which hyperfine parameters of the nitrite nitrogen were estimated. The small isotropic hyperfine coupling, 0.36 MHz, of the nitrite nitrogen (14N) suggests that the substrate binds in an axial position to the copper in the type 2 site and that the molecular orbital containing the unpaired electron extends onto the substrate. This and other changes in the EPR parameters occurring after nitrite binding suggest a change in electronic structure of the site, which most likely prepares the site for the catalytic reaction. We propose that this change is essential for the reaction to occur.

Research paper thumbnail of Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2005

Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types ... more Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type 1 is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes ...

Research paper thumbnail of Computational Library Design for Increasing Haloalkane Dehalogenase Stability

ChemBioChem, 2014

We explored the use of a computational design framework for the stabilization of the haloalkane d... more We explored the use of a computational design framework for the stabilization of the haloalkane dehalogenase LinB. Energy calculations, disulfide bond design, molecular dynamics simulations, and rational inspection of mutant structures predicted many stabilizing mutations. Screening of these in small mutant libraries led to the discovery of seventeen point mutations and one disulfide bond that enhanced thermostability. Mutations located in or contacting flexible regions of the protein had a larger stabilizing effect than mutations outside such regions. The combined introduction of twelve stabilizing mutations resulted in a LinB mutant with a 23 °C increase in apparent melting temperature (Tm,app , 72.5 °C) and an over 200-fold longer half-life at 60 °C. The most stable LinB variants also displayed increased compatibility with co-solvents, thus allowing substrate conversion and kinetic resolution at much higher concentrations than with the wild-type enzyme.

Research paper thumbnail of A Rearranging Ligand Enables Allosteric Control of Catalytic Activity in Copper-containing Nitrite Reductase

Journal of Molecular Biology, 2006

In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of t... more In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of the type-1 site was replaced (M150G) to make the copper ion accessible to external ligands that might affect the enzyme's catalytic activity. The type-1 site optical spectrum of M150G (A 460 /A 600 Z 0.71) differs significantly from that of the native nitrite reductase (A 460 / A 600 Z1.3). The midpoint potential of the type-1 site of nitrite reductase M150G (E M Z312(G5) mV versus hydrogen) is higher than that of the native enzyme (E M Z213(G5) mV). M150G has a lower catalytic activity (k cat Z 133(G6) s K1 ) than the wild-type nitrite reductase (k cat Z416(G10) s K1 ). The binding of external ligands to M150G restores spectral properties, midpoint potential (E M !225 mV), and catalytic activity (k cat Z374(G28) s K1 ). Also the M150H (A 460 /A 600 Z7.7, E M Z104(G5) mV, k cat Z0.099(G0.006) s K1 ) and M150T (A 460 /A 600 Z0.085, E M Z340(G5) mV, k cat Z126(G2) s K1 ) variants were characterized. Crystal structures show that the ligands act as allosteric effectors by displacing Met62, which moves to bind to the Cu in the position emptied by the M150G mutation. The reconstituted type-1 site has an otherwise unaltered geometry. The observation that removal of an endogenous ligand can introduce allosteric control in a redox enzyme suggests potential for structural and functional flexibility of coppercontaining redox sites.

Research paper thumbnail of Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase

Biochimica Et Biophysica Acta Proteins and Proteomics, Aug 1, 2005

Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types ... more Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type 1 is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes ...

Research paper thumbnail of Computationally efficient and accurate enantioselectivity modeling by clusters of molecular dynamics simulations

Journal of chemical information and modeling, Jan 28, 2014

Computational approaches could decrease the need for the laborious high-throughput experimental s... more Computational approaches could decrease the need for the laborious high-throughput experimental screening that is often required to improve enzymes by mutagenesis. Here, we report that using multiple short molecular dynamics (MD) simulations makes it possible to accurately model enantioselectivity for large numbers of enzyme-substrate combinations at low computational costs. We chose four different haloalkane dehalogenases as model systems because of the availability of a large set of experimental data on the enantioselective conversion of 45 different substrates. To model the enantioselectivity, we quantified the frequency of occurrence of catalytically productive conformations (near attack conformations) for pairs of enantiomers during MD simulations. We found that the angle of nucleophilic attack that leads to carbon-halogen bond cleavage was a critical variable that limited the occurrence of productive conformations; enantiomers for which this angle reached values close to 180° ...

Research paper thumbnail of Enantioselective Enzymes by Computational Design and In Silico Screening

Angewandte Chemie (International ed. in English), Jan 4, 2015

Computational enzyme design holds great promise for providing new biocatalysts for synthetic chem... more Computational enzyme design holds great promise for providing new biocatalysts for synthetic chemistry. A strategy to design small mutant libraries of complementary enantioselective epoxide hydrolase variants for the production of highly enantioenriched (S,S)-diols and (R,R)-diols is developed. Key features of this strategy (CASCO, catalytic selectivity by computational design) are the design of mutations that favor binding of the substrate in a predefined orientation, the introduction of steric hindrance to prevent unwanted substrate binding modes, and ranking of designs by high-throughput molecular dynamics simulations. Using this strategy we obtained highly stereoselective mutants of limonene epoxide hydrolase after experimental screening of only 37 variants. The results indicate that computational methods can replace a substantial amount of laboratory work when developing enantioselective enzymes.

Research paper thumbnail of Stabilization of cyclohexanone monooxygenase by a computationally designed disulfide bond spanning only one residue

FEBS Open Bio, 2014

Enzyme stability is an important parameter in biocatalytic applications, and there is a strong ne... more Enzyme stability is an important parameter in biocatalytic applications, and there is a strong need for efficient methods to generate robust enzymes. We investigated whether stabilizing disulfide bonds can be computationally designed based on a model structure. In our approach, unlike in previous disulfide engineering studies, short bonds spanning only a few residues were included. We used cyclohexanone monooxygenase (CHMO), a Baeyer-Villiger monooxygenase (BVMO) from Acinetobacter sp. NCIMB9871 as the target enzyme. This enzyme has been the prototype BVMO for many biocatalytic studies even though it is notoriously labile. After creating a small library of mutant enzymes with introduced cysteine pairs and subsequent screening for improved thermostability, three stabilizing disulfide bonds were identified. The introduced disulfide bonds are all within 12 Å of each other, suggesting this particular region is critical for unfolding. This study shows that stabilizing disulfide bonds do not have to span many residues, as the most stabilizing disulfide bond, L323C-A325C, spans only one residue while it stabilizes the enzyme, as shown by a 6°C increase in its apparent melting temperature.

Research paper thumbnail of Computational design gains momentum in enzyme catalysis engineering

FEBS Journal, 2013

Computational protein design is becoming a powerful tool for tailoring enzymes for specific biote... more Computational protein design is becoming a powerful tool for tailoring enzymes for specific biotechnological applications. When applied to existing enzymes, computational re-design makes it possible to obtain orders of magnitude improvement in catalytic activity towards a new target substrate. Computational methods also allow the design of completely new active sites that catalyze reactions that are not known to occur in biological systems. If initial designs display modest catalytic activity, which is often the case, this may be improved by iterative cycles of computational design or by follow-up engineering through directed evolution. Compared to established protein engineering methods such as directed evolution and structure-based mutagenesis, computational design allows for much larger jumps in sequence space; for example, by introducing more than a dozen mutations in a single step or by introducing loops that provide new functional interactions. Recent advances in the computational design toolbox, which include new backbone re-design methods and the use of molecular dynamics simulations to better predict the catalytic activity of designed variants, will further enhance the use of computational tools in enzyme engineering.

Research paper thumbnail of Computationally designed libraries for rapid enzyme stabilization

Protein Engineering Design and Selection, 2014

The ability to engineer enzymes and other proteins to any desired stability would have wide-rangi... more The ability to engineer enzymes and other proteins to any desired stability would have wide-ranging applications.

Research paper thumbnail of Protein Film Voltammetry of Copper-Containing Nitrite Reductase Reveals Reversible Inactivation

Journal of the American Chemical Society, 2007

The Cu-containing nitrite reductase from Alcaligenes faecalis S-6 catalyzes the one-electron redu... more The Cu-containing nitrite reductase from Alcaligenes faecalis S-6 catalyzes the one-electron reduction of nitrite to nitric oxide (NO). Electrons enter the enzyme at the so-called type-1 Cu site and are then transferred internally to the catalytic type-2 Cu site. Protein film voltammetry experiments were carried out to obtain detailed information about the catalytic cycle. The homotrimeric structure of the enzyme is reflected in a distribution of the heterogeneous electron-transfer rates around three main values. Otherwise, the properties and the mode of operation of the enzyme when it is adsorbed as a film on a pyrolytic graphite electrode are essentially unchanged compared to those of the free enzyme in solution. It was established that the reduced type-2 site exists in either an active or an inactive conformation with an interconversion rate of approximately 0.1 s(-1). The random sequential mechanism comprises two routes, one in which the type-2 site is reduced first and subsequently binds nitrite, which is then converted into NO, and another in which the oxidized type-2 site binds nitrite and then accepts an electron to produce NO. At high nitrite concentration, the second route prevails and internal electron transfer is rate-limiting. The midpoint potentials of both sites could be established under catalytic conditions. Binding of nitrite to the type-2 site does not affect the midpoint potential of the type-1 site, thereby excluding cooperativity between the two sites.

Research paper thumbnail of Effect of the Methionine Ligand on the Reorganization Energy of the Type-1 Copper Site of Nitrite Reductase

Journal of the American Chemical Society, 2007

Copper-containing nitrite reductase harbors a type-1 and a type-2 Cu site. The former acts as the... more Copper-containing nitrite reductase harbors a type-1 and a type-2 Cu site. The former acts as the electron acceptor site of the enzyme, and the latter is the site of catalytic action. The effect of the methionine ligand on the reorganization energy of the type-1 site was explored by studying the electrontransfer kinetics between NiR (wild type (wt) and the variants Met150Gly and Met150Thr) with Fe(II)EDTA and Fe(II)HEDTA. The mutations increased the reorganization energy by 0.3 eV (30 kJ mol -1 ). A similar increase was found from pulse radiolysis experiments on the wt NIR and three variants (Met150Gly, Met150His, and Met150Thr). Binding of the nearby Met62 to the type-1 Cu site in Met150Gly (under influence of an allosteric effector) lowered the reorganization energy back to approximately the wt value. According to XRD data the structure of the reduced type-1 site in Met150Gly NiR in the presence of an allosteric effector is similar to that in the reduced wt NiR (solved to 1.85 Å), compatible with the similarity in reorganization energy.

Research paper thumbnail of A Rearranging Ligand Enables Allosteric Control of Catalytic Activity in Copper-containing Nitrite Reductase

Journal of Molecular Biology, 2006

In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of t... more In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of the type-1 site was replaced (M150G) to make the copper ion accessible to external ligands that might affect the enzyme's catalytic activity. The type-1 site optical spectrum of M150G (A(460)/A(600)=0.71) differs significantly from that of the native nitrite reductase (A(460)/A(600)=1.3). The midpoint potential of the type-1 site of nitrite reductase M150G (E(M)=312(+/-5)mV versus hydrogen) is higher than that of the native enzyme (E(M)=213(+/-5)mV). M150G has a lower catalytic activity (k(cat)=133(+/-6)s(-1)) than the wild-type nitrite reductase (k(cat)=416(+/-10)s(-1)). The binding of external ligands to M150G restores spectral properties, midpoint potential (E(M)<225mV), and catalytic activity (k(cat)=374(+/-28)s(-1)). Also the M150H (A(460)/A(600)=7.7, E(M)=104(+/-5)mV, k(cat)=0.099(+/-0.006)s(-1)) and M150T (A(460)/A(600)=0.085, E(M)=340(+/-5)mV, k(cat)=126(+/-2)s(-1)) variants were characterized. Crystal structures show that the ligands act as allosteric effectors by displacing Met62, which moves to bind to the Cu in the position emptied by the M150G mutation. The reconstituted type-1 site has an otherwise unaltered geometry. The observation that removal of an endogenous ligand can introduce allosteric control in a redox enzyme suggests potential for structural and functional flexibility of copper-containing redox sites.

Research paper thumbnail of A Random-sequential Mechanism for Nitrite Binding and Active Site Reduction in Copper-containing Nitrite Reductase

Journal of Biological Chemistry, 2006

The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 cop... more The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of pH and nitrite on the turnover rate in the presence of three different electron donors at saturating concentrations were measured. The activity of NiR was also measured electrochemically by exploiting direct electron transfer to the enzyme immobilized on a graphite rotating disk electrode. In all cases, the steady-state kinetics fitted excellently to a random-sequential mechanism in which electron transfer from the type-1 to the type-2 site is rate-limiting. At low [NO(-)(2)] reduction of the type-2 site precedes nitrite binding, at high [NO(-)(2)] the reverse occurs. Below pH 6.5, the catalytic activity diminished at higher nitrite concentrations, in agreement with electron transfer being slower to the nitrite-bound type-2 site than to the water-bound type-2 site. Above pH 6.5, substrate activation is observed, in agreement with electron transfer to the nitrite-bound type-2 site being faster than electron transfer to the hydroxyl-bound type-2 site. To study the effect of slower electron transfer between the type-1 and type-2 site, NiR M150T was used. It has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an approximately 50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism.

Research paper thumbnail of Aminoacyl-coenzyme A synthesis catalyzed by a CoA ligase from Penicillium chrysogenum

FEBS Letters, 2011

Keywords: Coenzyme A ligase Aminoacyl-coenzyme A b-Phenylalanine Penicillium chrysogenum a b s t ... more Keywords: Coenzyme A ligase Aminoacyl-coenzyme A b-Phenylalanine Penicillium chrysogenum a b s t r a c t Coenzyme A ligases play an important role in metabolism by catalyzing the activation of carboxylic acids. In this study we describe the synthesis of aminoacyl-coenzyme As (CoAs) catalyzed by a CoA ligase from Penicillium chrysogenum. The enzyme accepted medium-chain length fatty acids as the best substrates, but the proteinogenic amino acids L-phenylalanine and L-tyrosine, as well as the non-proteinogenic amino acids D-phenylalanine, D-tyrosine and (R)-and (S)-b-phenylalanine were also accepted. Of these amino acids, the highest activity was found for (R)-b-phenylalanine, forming (R)-b-phenylalanyl-CoA. Homology modeling suggested that alanine 312 is part of the active site cavity, and mutagenesis (A312G) yielded a variant that has an enhanced catalytic efficiency with b-phenylalanines and D-a-phenylalanine.

Research paper thumbnail of Thermal stability effects of removing the type-2 copper ligand His306 at the interface of nitrite reductase subunits

European Biophysics Journal, 2007

Nitrite reductase (NiR) is a highly stable trimeric protein, which denatures via an intermediate,... more Nitrite reductase (NiR) is a highly stable trimeric protein, which denatures via an intermediate, U-unfolded and F-final). To understand the role of interfacial residues on protein stability, a type-2 copper site ligand, His306, has been mutated to an alanine. The characterization of the native state of the mutated protein highlights that this mutation prevents copper ions from binding to the type-2 site and eliminates catalytic activity. No significant alteration of the geometry of the type-1 site is observed. Study of the thermal denaturation of this His306Ala NiR variant by differential scanning calorimetry shows an endothermic irreversible profile, with maximum heat absorption at T max % 85°C, i.e., 15°C lower than the corresponding value found for wild-type protein. The reduction of the protein thermal stability induced by the His306Ala replacement was also shown by optical spectroscopy. The denaturation pathway of the variant is compatible with the kinetic model N 3 ! k F 3 ; where the protein irreversibly passes from the native to the final state. No evidence of subunits' dissociation has been found within the unfolding process. The results show that the type-2 copper sites, situated at the interface of two monomers, significantly contribute to both the stability and the denaturation mechanism of NiR.

Research paper thumbnail of Structure- and sequence-analysis inspired engineering of proteins for enhanced thermostability

Current Opinion in Structural Biology, 2013

Protein engineering strategies for increasing stability can be improved by replacing random mutag... more Protein engineering strategies for increasing stability can be improved by replacing random mutagenesis and high-throughput screening by approaches that include bioinformatics and computational design. Mutations can be focused on regions in the structure that are most flexible and involved in the early steps of thermal unfolding. Sequence analysis can often predict the position and nature of stabilizing mutations, and may allow the reconstruction of thermostable ancestral sequences. Various computational tools make it possible to design stabilizing features, such as hydrophobic clusters and surface charges. Different methods for designing chimeric enzymes can also support the engineering of more stable proteins without the need of high-throughput screening.

Research paper thumbnail of Directed Evolution Strategies for Enantiocomplementary Haloalkane Dehalogenases: From Chemical Waste to Enantiopure Building Blocks

Research paper thumbnail of Bidirectional Catalysis by Copper-Containing Nitrite Reductase †

Biochemistry, 2004

The copper-containing nitrite reductase from Alcaligenes faecalis S-6 was found to catalyze the o... more The copper-containing nitrite reductase from Alcaligenes faecalis S-6 was found to catalyze the oxidation of nitric oxide to nitrite, the reverse of its physiological reaction. Thermodynamic and kinetic constants with the physiological electron donor pseudoazurin were determined for both directions of the catalyzed reaction in the pH range of 6-8. For this, nitric oxide was monitored by a Clark-type electrode, and the redox state of pseudoazurin was measured by optical spectroscopy. The equilibrium constant (K(eq)) depends on the reduction potentials of pseudoazurin and nitrite/nitric oxide, both of which vary with pH. Above pH 6.2 the formation of NiR substrates (nitrite and reduced pseudoazurin) is favored over the products (NO and oxidized pseudoazurin). At pH 8 the K(eq) amounts to 10(3). The results show that dissimilatory nitrite reductases catalyze an unfavorable reaction at physiological pH (pH = 7-8). Consequently, nitrous oxide production by copper-containing nitrite reductases is unlikely to occur in vivo with a native electron donor. With increasing pH, the rate and specificity constant of the forward reaction decrease and become lower than the rate of the reverse reaction. The opposite occurs for the rate of the reverse reaction; thus the catalytic bias for nitrite reduction decreases. At pH 6.0 the k(cat) for nitrite reduction was determined to be 1.5 x 10(3) s(-1), and at pH 8 the rate of the reverse reaction is 125 s(-1).

Research paper thumbnail of Reconstitution of the Type-1 Active Site of the H145G/A Variants of Nitrite Reductase by Ligand Insertion †

Biochemistry, 2003

Variants of the copper-containing nitrite reductase (NiR) of Alcaligenes faecalis S6 were constru... more Variants of the copper-containing nitrite reductase (NiR) of Alcaligenes faecalis S6 were constructed by site-directed mutagenesis, by which the C-terminal histidine ligand (His145) of the Cu in the type-1 site was replaced by an alanine or a glycine. The type-1 sites in the NiR variants as isolated, are in the reduced form, but can be oxidized in the presence of external ligands, like (substituted) imidazoles and chloride. The reduction potential of the type-1 site of NiR-H145A reconstituted with imidazole amounts to 505 mV vs NHE (20 degrees C, pH 7, 10 mM imidazole), while for the native type-1 site it amounts to 260 mV. XRD data on crystals of the reduced and oxidized NiR-H145A variant show that in the reduced type-1 site the metal is 3-coordinated, but in the oxidized form takes up a ligand from the solution. With the fourth (exogenous) ligand in place the type-1 site is able to accept electrons at about the same rate as the wt NiR, but it is unable to pass the electron onto the type-2 site, leading to loss of enzymatic activity. It is argued that the uptake of an electron by the mutated type-1 site is accompanied by a loss of the exogenous ligand and a concomitant rise of the redox potential. This rise effectively traps the electron in the type-1 site.

Research paper thumbnail of The Substrate-Bound Type 2 Copper Site of Nitrite Reductase:  The Nitrogen Hyperfine Coupling of Nitrite Revealed by Pulsed EPR †

Biochemistry, 2005

A pulsed electron paramagnetic resonance study has been performed on the type 2 copper site of ni... more A pulsed electron paramagnetic resonance study has been performed on the type 2 copper site of nitrite reductase (NiR) from Alcaligenes faecalis. The H145A mutant, in which histidine 145 is replaced by alanine, was studied by ESEEM and HYSCORE experiments at 9 GHz on frozen solutions. This mutant contains a reduced type 1 copper site which allowed a selective investigation of the type 2 site of H145A and of its nitrite-bound form H145A (NO2(-)). The experiments yielded hyperfine and quadrupole parameters of the remote nitrogens of two of the histidines in the type 2 copper site of the protein and revealed the changes of these values induced by substrate binding (14NO2(-) and 15NO2(-)). The HYSCORE experiments displayed a signal of 15NO2(-) bound to H145A, from which hyperfine parameters of the nitrite nitrogen were estimated. The small isotropic hyperfine coupling, 0.36 MHz, of the nitrite nitrogen (14N) suggests that the substrate binds in an axial position to the copper in the type 2 site and that the molecular orbital containing the unpaired electron extends onto the substrate. This and other changes in the EPR parameters occurring after nitrite binding suggest a change in electronic structure of the site, which most likely prepares the site for the catalytic reaction. We propose that this change is essential for the reaction to occur.

Research paper thumbnail of Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2005

Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types ... more Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type 1 is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes ...