Jens Erik Nielsen

Jens Erik Nielsen
Novozymes · Enzyme Research

PhD

About

72
Publications
14,048
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11,231
Citations
Additional affiliations
May 2017 - October 2017
Novozymes
Position
  • Manager
August 2011 - April 2016
Novozymes
Position
  • Manager
September 2003 - August 2011
University College Dublin

Publications

Publications (72)
Article
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α-L-Arabinofuranosidases from glycoside hydrolase family 51 use a stereochemically retaining hydrolytic mechanism to liberate nonreducing terminal α-L-arabinofuranose residues from plant polysaccharides such as arabinoxylan and arabinan. To date, more than ten fungal GH51 α-L-arabinofuranosidases have been functionally characterized, yet no structu...
Article
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Background Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy...
Article
We have characterized the structure and dynamics of the carbohydrate-modifying enzyme Paenibacillus nanensis xanthan lyase (PXL) involved in the degradation of xanthan by X-ray crystallography, small-angle X-ray scattering, and hydrogen/deuterium exchange mass spectrometry. Unlike other xanthan lyases, PXL is specific for both unmodified mannose an...
Article
The precise catalytic strategies used for the breakdown of the complex bacterial polysaccharide xanthan, an increasingly frequent component of processed human foodstuffs, have remained a mystery. Here we present the characterization of an endo-xanthanase from Paenibacillus sp. 62047. We show that it is a CAZy family 9 glycoside hydrolase (GH9) resp...
Article
The Adaptive Poisson-Boltzmann Solver (APBS) software was developed to solve the equations of continuum electrostatics for large biomolecular assemblages that has provided impact in the study of a broad range of chemical, biological, and biomedical applications. APBS addresses three key technology challenges for understanding solvation and electros...
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Full-text available
GH-18 chitinases are chitinolytic enzymes, primarily responsible for the recycling of insoluble chitin biomaterials. These enzymes contain three invariant acidic active-site residues within a DXDXE motif, which play a synergistic role in the catalytic cycle of chitin degradation. We employed a pKa calculation approach to approximate the protonation...
Article
A microcrystalline suspension of Bacillus lentus subtilisin (Savinase) produced during industrial large-scale production was analysed by X-ray powder diffraction (XRPD) and X-ray single-crystal diffraction (MX). XRPD established that the bulk microcrystal sample representative of the entire production suspension corresponded to space group P212121,...
Article
Understanding the connection between protein structure and function requires a quantitative understanding of electrostatic effects. Structure-based electrostatics calculations are essential for this purpose, but their use have been limited by a long-standing discussion on which value to use for the dielectric constants (εeff and εp) required in Cou...
Article
a b s t r a c t The coupled effect of ionic strength (50–400 mM) and pH (2–8) on ionization and conformation equilibria of lysozyme was studied using NMR spectroscopy. Observed changes in pK a values of the ionizable groups were found to originate from perturbations in the geometry of hydrogen bonds rather than screening of electric fields. Moreove...
Article
The pH-dependent activity of wild-type Bacillus circulans xylanase (BcX) is set by the pKa values of its nucleophile Glu78 and general acid/base Glu172. Herein, we examined several strategies to manipulate these pKa values and thereby shift the pHopt at which BcX is optimally active. Altering the global charge of BcX through random succinylation ha...
Article
Raw data from experiments across the biological sciences comes in a large variety of text formats. In small or medium sized laboratories researchers often use an assorted collection of software to interpret, fit, and visualize their data. The spreadsheet is commonly the core component of such a workflow. The limitations of such programs for large a...
Article
The stability of serine proteases is of major importance for their application in industrial processes. Here we study the determinants of the stability of a Nocardiopsis prasina serine protease using fast residual activity assays, a feature classification algorithm, and structure-based energy calculation algorithms for 121 micropurified mutant enzy...
Article
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The exchange of information between experimentalists and theoreticians is crucial to improving the predictive ability of theoretical methods and hence our understanding of the related biology. However many barriers exist which prevent the flow of information between the two disciplines. Enabling effective collaboration requires that experimentalist...
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The majority of pKa values in protein unfolded states are close to the amino acid model pKa values, thus reflecting the weak intramolecular interactions present in the unfolded ensemble of most proteins. We have carried out thermal denaturation measurements on the WT and eight mutants of HEWL from pH 1.5 to pH 11.0 to examine the unfolded state pKa...
Article
Protein kinases (PK) control phosphorylation in eukaryotic cells, and thereby regulate metabolic pathways, cell cycle progression, apoptosis, and transcription. Consequently, there is significant interest in manipulating PK activity and treat diseases by using small-molecule drugs. All PK catalytic domains undergo large conformational changes as a...
Article
A central aim of computational biology is the prediction of experimentally observable biophysical characteristics of proteins. In the past decade, a large number of tools have been developed for predicting the effect of single-point mutations on protein stability, driven in part by the large amount of experimental data available for this phenomenon...
Article
Protein pK(a) calculation methods are developed partly to provide fast non-experimental estimates of the ionization constants of protein side chains. However, the most significant reason for developing such methods is that a good pK(a) calculation method is presumed to provide an accurate physical model of protein electrostatics, which can be appli...
Article
Microphthalmia, anophthalmia, and coloboma (MAC) are structural congenital eye malformations that cause a significant proportion of childhood visual impairments. Several disease genes have been identified but do not account for all MAC cases, suggesting that additional risk loci exist. We used single nucleotide polymorphism (SNP) homozygosity mappi...
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The pK(a) -cooperative aims to provide a forum for experimental and theoretical researchers interested in protein pK(a) values and protein electrostatics in general. The first round of the pK(a) -cooperative, which challenged computational labs to carry out blind predictions against pK(a) s experimentally determined in the laboratory of Bertrand Ga...
Article
The pK(a) Cooperative (http://www.pkacoop.org) was organized to advance development of accurate and useful computational methods for structure-based calculation of pK(a) values and electrostatic energies in proteins. The Cooperative brings together laboratories with expertise and interest in theoretical, computational, and experimental studies of p...
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NMR-monitored pH titration curves of proteins provide a rich source of structural and electrostatic information. Although relatively straightforward to measure, interpreting pH-dependent chemical shift changes to obtain site-specific acid dissociation constants (pK A values) is challenging. In order to analyze the biphasic titrations exhibited by t...
Article
Although important shifts in the isoelectric point of prokaryotic proteins, mainly due to adaptation to environmental pH, have been widely reported, such studies have not covered mammalian proteins, where pH changes may relate to changes in subcellular or tissue compartmentalization. We explored the isoelectric point of the proteome of 13 mammalian...
Article
APBS and PDB2PQR are widely utilized free software packages for biomolecular electrostatics calculations. Using the Opal toolkit, we have developed a Web services framework for these software packages that enables the use of APBS and PDB2PQR by users who do not have local access to the necessary amount of computational capabilities. This not only i...
Article
Full-text available
Electrostatic forces play a large role in determining the strength of protein-ligand interactions, and the calculation of pKa value shifts upon ligand binding is therefore an important component of any accurate protein-ligand binding calculation. However, such pKa calculations are rarely performed in virtual screening experiments due to the unavail...
Article
Site-specific pK(a) values measured by NMR spectroscopy provide essential information on protein electrostatics, the pH-dependence of protein structure, dynamics and function, and constitute an important benchmark for protein pK(a) calculation algorithms. Titration curves can be measured by tracking the NMR chemical shifts of several reporter nucle...
Article
Site-directed mutagenesis is routinely used in modern biology to elucidate the functional or biophysical roles of protein residues, and plays an important role in the field of rational protein design. Over the past decade, a number of computational tools have been developed that can predict the effect of point mutations on a protein's biophysical c...
Article
The conformational change observed upon ligand binding and phosphorylation for the cAMP-dependent protein kinase (protein kinase A-PKA) is of high importance for the regulation of its activity. We calculate pKa values and net charges for 18 3D structures of PKA in various conformations and liganded states to examine the role of electrostatics in li...
Article
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Large amounts of data are being generated annually on the connection between the sequence, structure and function of proteins using site-directed mutagenesis, protein design and directed evolution techniques. These data provide the fundamental building blocks for our understanding of protein function, molecular biology and living organisms in gener...
Article
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Accurate computational methods for predicting electrostatic energies are of major importance for our understanding of protein energetics in general for computer-aided drug design as well as for the design of novel biocatalysts and protein therapeutics. Electrostatic energies are of particular importance in such applications as virtual screening, dr...
Article
pH-induced chemical shift perturbations (CSPs) can be used to study pH-dependent conformational transitions in proteins. Recently, an elegant principal component analysis (PCA) algorithm was developed and used to study the pH-dependent structural transitions in bovine beta-lactoglobulin (betaLG) by analyzing its NMR pH-titration spectra. Here, we a...
Article
Full-text available
NMR-monitored pH titration experiments are routinely used to measure site-specific protein pKa values. Accurate experimental pKa values are essential in dissecting enzyme catalysis, in studying the pH-dependence of protein stability and ligand binding, in benchmarking pKa prediction algorithms, and ultimately in understanding electrostatic effects...
Article
Full-text available
The glycosaminoglycan heparin is known to possess antimetastatic activity in experimental models and preclinical studies, but there is still uncertainty over its mechanism of action in this respect. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III, but a similar cofactor role has not been previously invest...
Article
The development of docking scoring functions requires high-resolution 3D structures of protein-ligand complexes for which the binding affinity of the ligand has been measured experimentally. Protein-ligand binding affinities are measured in solution experiments, and high resolution protein-ligand structures can be determined only by X-ray crystallo...
Article
Full-text available
Hyaluronidases from diverse species and sources have different pH optima. Distinct mechanisms with regard to dynamic structural changes, which control hyaluronidase activity at varying pH, are unknown. Human serum hyaluronidase 1 (HYAL1) is active solely below pH 5.1. Here we report the design of a HYAL1 variant that degrades hyaluronan up to pH 5....
Article
The pH dependence of protein biophysical characteristics is often analyzed to gain an improved understanding of protein stability, enzyme activity, and protein-ligand-binding processes. Indeed, much of our understanding of the catalytic mechanisms of enzymes derives from studies of the pH dependence of catalytic activity, and the ability to redesig...
Article
The pH-dependence of the NMR chemical shift for protein residues provides a wealth of information on biophysical characteristics important for the function of proteins. It is well known that pKa values can be extracted from protein NMR pH-titration curves, but recent work has illustrated that NMR titration curves also can give information on pair w...
Article
The pH-dependence of the NMR chemical shift for titratable groups in proteins often deviate from a standard Henderson-Hasselbalch (HH) titration curve. A non-HH dependence of the chemical shift for a given residue can arise from a single-site, non-HH titrational event for that residue, or if the chemical shift of the group is influenced by addition...
Article
Full-text available
Real-world observable physical and chemical characteristics are increasingly being calculated from the 3D structures of biomolecules. Methods for calculating pKa values, binding constants of ligands, and changes in protein stability are readily available, but often the limiting step in computational biology is the conversion of PDB structures into...
Article
We have characterized by NMR spectroscopy the three active site (His80, His85, and His205) and two non-active site (His107 and His114) histidines in the 34 kDa catalytic domain of Cellulomonas fimi xylanase Cex in its apo, noncovalently aza-sugar-inhibited, and trapped glycosyl-enzyme intermediate states. Due to protection from hydrogen exchange, t...
Article
The ability to re-engineer enzymatic pH-activity profiles is of importance for industrial applications of enzymes. We theoretically explore the feasibility of re-engineering enzymatic pH-activity profiles by changing active site pK(a) values using point mutations. We calculate the maximum achievable DeltapK(a) values for 141 target titratable group...
Article
The results of protein pKa calculations are routinely being analysed to understand the pH-dependence of protein characteristics such as stability and catalysis. Systems of functionally important titratable groups are identified from protein from pKa calculations, but the rationalisation of the behaviour of such systems is inherently problematic due...
Article
pK(a) calculations for macromolecules are normally performed by solving the Poisson-Boltzmann equation, accounting for the different dielectric constants of solvent and solute, as well as the ionic strength. Despite the large number of successful applications, there are some situations where the current algorithms are not suitable: (1) large scale,...
Article
Full-text available
Hypoxia is a feature of the microenvironment of a growing tumor. The transcription factor NFκB is activated in hypoxia, an event that has significant implications for tumor progression. Here, we demonstrate that hypoxia activates NFκB through a pathway involving activation of IκB kinase-β (IKKβ) leading to phosphorylation-dependent degradation of I...
Article
Hypoxia is a feature of the microenvironment of a growing tumor. The transcription factor NFkappaB is activated in hypoxia, an event that has significant implications for tumor progression. Here, we demonstrate that hypoxia activates NFkappaB through a pathway involving activation of IkappaB kinase-beta (IKKbeta) leading to phosphorylation-dependen...
Article
The retromer complex is involved in the retrograde transport of the CI-M6PR (cation-independent mannose 6-phosphate receptor) from endosomes to the Golgi. It is a hetero-trimeric complex composed of Vps26 (vacuolar sorting protein 26), Vps29 and Vps35 proteins, which are conserved in eukaryote evolution. Recently, elucidation of the crystal structu...
Article
Full-text available
The pKa values in proteins govern the pH-dependence of protein stability and enzymatic activity. A large number of mutagenesis experiments have been carried out in the last three decades to re-engineer the pH-activity and pH-stability profile of enzymes and proteins. We have developed the pKD webserver (http://polymerase.ucd.ie/pKa_Design), which p...
Article
In this paper we present a method for the multi-resolution comparison of biomolecular electrostatic potentials without the need for global structural alignment of the biomolecules. The underlying computational geometry algorithm uses multi-resolution attributed contour trees (MACTs) to compare the topological features of volumetric scalar fields. W...
Article
Ubiquitin-conjugating enzymes (E2s or Ubcs) are essential components in the ubiquitination apparatus. These enzymes accept ubiquitin from an E1 enzyme and then, usually with the aid of an E3 enzyme, donate the ubiquitin to the target protein. The function of E2 relies critically on the chemistry of its active site cysteine residue since this residu...
Article
Continuum solvation models, such as Poisson–Boltzmann and Generalized Born methods, have become increasingly popular tools for investigating the influence of electrostatics on biomolecular structure, energetics and dynamics. However, the use of such methods requires accurate and complete structural data as well as force field parameters such as ato...
Article
Glycoside hydrolysis by retaining family 18 chitinases involves a catalytic acid (Glu) which is part of a conserved DXDXE sequence motif that spans strand four of a (betaalpha)8 barrel (TIM barrel) structure. These glycoside hydrolases are unusual in that the positive charge emerging on the anomeric carbon after departure of the leaving group is st...
Article
The ionization properties of the active-site residues in enzymes are of considerable interest in the study of the catalytic mechanisms of enzymes. Knowledge of these ionization constants (pKa values) often allows the researcher to identify the proton donor and the catalytic nucleophile in the reaction mechanism of the enzyme. Estimates of protein r...
Article
The calculation of the physical properties of a protein from its X-ray structure is of importance in virtually every aspect of modern biology. Although computational algorithms have been developed for calculating everything from the dynamics of a protein to its binding specificity, only limited information is available on the ability of these metho...
Article
Rational engineering of enzyme properties has an enormous potential but is hampered by limitations in our understanding of the structural determinants of these properties. Recent examples of efforts in rational engineering illustrate the current state of the art.
Article
We have developed a computer algorithm, FOLDEF (for FOLD-X energy function), to provide a fast and quantitative estimation of the importance of the interactions contributing to the stability of proteins and protein complexes. The predictive power of FOLDEF was tested on a very large set of point mutants (1088 mutants) spanning most of the structura...
Article
Full-text available
The impact of long range electrostatic interactions on catalysis in the thermolysin-like protease from Bacillus stearothermophilus was studied by analyzing the effects of inserting or removing charges on the protein surface. Various mutations were introduced at six different positions, and double-mutant cycle analysis was used to study the extent t...
Article
Previous studies of the low molecular mass family 11 xylanase from Bacillus circulans show that the ionization state of the nucleophile (Glu78, pKa 4.6) and the acid/base catalyst (Glu172, pKa 6.7) gives rise to its pH-dependent activity profile. Inspection of the crystal structure of BCX reveals that Glu78 and Glu172 are in very similar environmen...
Article
The glycosyl hydrolases present a large family of enzymes that are of great significance for industry. Consequently, there is considerable interest in engineering the enzymes in this family for optimal performance under a range of very diverse conditions. Until recently, tailoring glycosyl hydrolases for specific industrial processes mainly involve...
Article
Full-text available
The glycosyl hydrolases present a large family of enzymes that are of great significance for industry. Consequently, there is considerable interest in engineering the enzymes in this family for optimal performance under a range of very diverse conditions. Until recently, tailoring glycosyl hydrolases for specific industrial processes mainly involve...
Article
pK(a) calculation methods that are based on finite difference solutions to the Poisson-Boltzmann equation (FDPB) require that energy calculations be performed for a large number of different protonation states of the protein. Normally, the differences between these protonation states are modeled by changing the charges on a few atoms, sometimes the...
Article
alpha-Amylases constitute a very diverse family of glycosyl hydrolases that cleave alpha1-->4 linkages in amylose and related polymers. Recent structural and mutagenic studies of archeael, mammalian and bacterial alpha-amylases have resulted in a wealth of information on the catalytic mechanism and on the structural features of this enzyme class. B...
Article
The amino acid sequence of Leishmania mexicana triose phosphate isomerase is unique in having at position 65 a glutamic acid instead of a glutamine. The stability properties of LmTIM and the E65Q mutant were investigated by pH and guanidinium chloride-induced unfolding. The crystal structure of E65Q was determined. Three important observations were...
Article
The importance of electrostatics in catalysis has been emphasized in the literature for a large number of enzymes. We examined this hypothesis for the Bacillus licheniformis alpha-amylase by constructing site-directed mutants that were predicted to change the pKa values of the catalytic residues and thus change the pH-activity profile of the enzyme...
Article
Electrostatic interactions play a key role in many aspects of protein engineering. Consequently, much effort has been put into the design of software for calculating electrostatic fields around macromolecules. We show that optimization of hydrogen bonding networks can improve both the results of pK(a) calculations and the results of electrostatic c...
Article
Full-text available
INTRODUCTION Hen Egg White Lysozyme, HEWL, is a well studied protein of 129 amino acids. The crystal structure was solved in 1965, and the folding pathway was elucidated in 1976. It has 60% sequence identity to human lysozyme, mutations of which are known to promote systemic amyloidosis under physiological conditions (1).

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