- A.E. Mars (1)
- A.J. Mulholland (2)
- W. Norde (2)
- J. Oost van der (3)
- S. Peter (1)
- L. Ridder (2)
- I.M.C.M. Rietjens (2)
- J.P.M. Sanders (1)
- B.P.L. Snijders (1)
- J. Springer (1)
- J.J.M. Vervoort (2)
- J. Vidal Sangra (1)
- M.J.G. Vos de (1)
- K.S. Vuoristo (1)
- J. Walther (1)
- H.J.G. Werken van de (1)
- R.A. Weusthuis (1)
- P.C. Wright (1)
Metabolic engineering of itaconate production in Escherichia coli
Vuoristo, K.S. ; Mars, A.E. ; Vidal Sangra, J. ; Springer, J. ; Eggink, G. ; Sanders, J.P.M. ; Weusthuis, R.A. - \ 2015
Applied Microbiology and Biotechnology 99 (2015)1. - ISSN 0175-7598 - p. 221 - 228.
aconitic acid decarboxylase - aspergillus-terreus - biotechnological production - acetate accumulation - citrate synthase - lactic-acid - fermentation - growth - expression - mutant
Interest in sustainable development has led to efforts to replace petrochemical-based monomers with biomass-based ones. Itaconic acid, a C5-dicarboxylic acid, is a potential monomer for the chemical industry with many prospective applications. cis-aconitate decarboxylase (CadA) is the key enzyme of itaconate production, converting the citric acid cycle intermediate cis-aconitate into itaconate. Heterologous expression of cadA from Aspergillus terreus in Escherichia coli resulted in low CadA activities and production of trace amounts of itaconate on Luria-Bertani (LB) medium (
Kinetically controlled refolding of a heat denatured hyperthermostable protein
Koutsopoulos, S. ; Oost, J. van der; Norde, W. - \ 2007
FEBS Journal 274 (2007)22. - ISSN 1742-464X - p. 5915 - 5923.
archaeon pyrococcus-furiosus - crystal-structure - thermodynamic properties - thermal-denaturation - citrate synthase - stability - temperature - enzyme - water - dehydrogenase
The thermal denaturation of endo-ß-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus was studied by calorimetry. The calorimetric profile revealed two transitions at 109 and 144¿°C, corresponding to protein denaturation and complete unfolding, respectively, as shown by circular dichroism and fluorescence spectroscopy data. Calorimetric studies also showed that the denatured state did not refold to the native state unless the cooling temperature rate was very slow. Furthermore, previously denatured protein samples gave well-resolved denaturation transition peaks and showed enzymatic activity after 3 and 9¿months of storage, indicating slow refolding to the native conformation over time.
Reconstruction of central carbon metabolism in Sulfolobus solfataricus using a two-dimensional gel electrophoresis map, stable isotope labelling and DNA microarray analysis
Snijders, B.P.L. ; Walther, J. ; Peter, S. ; Kinnman, I. ; Vos, M.J.G. de; Werken, H.J.G. van de; Brouns, S.J.J. ; Oost, J. van der; Wright, P.C. - \ 2006
Proteomics 6 (2006)5. - ISSN 1615-9853 - p. 1518 - 1529.
entner-doudoroff pathway - citric-acid cycle - hyperthermophilic archaea - archaebacterium sulfolobus - citrate synthase - phosphoenolpyruvate carboxylase - thermoacidophilic crenarchaeon - thermoplasma-acidophilum - thermoproteus-tenax - glycolytic pathways
In the last decade, an increasing number of sequenced archaeal genomes have become available, opening up the possibility for functional genomic analyses. Here, we reconstructed the central carbon metabolism in the hyperthermophilic crenarchaeon Sulfolobus solfataricus (glycolysis, gluconeogenesis and tricarboxylic acid cycle) on the basis of genomic, proteomic, transcriptomic and biochemical data. A 2-DE reference map of S. solfataricus grown on glucose, consisting of 325 unique ORFs in 255 protein spots, was created to facilitate this study. The map was then used for a differential expression study based on (15)N metabolic labelling (yeast extract + tryptone-grown cells (YT) vs. glucose-grown cells (G)). In addition, the expression ratio of the genes involved in carbon metabolism was studied using DNA microarrays. Surprisingly, only 3 and 14% of the genes and proteins, respectively, involved in central carbon metabolism showed a greater than two-fold change in expression level. All results are discussed in the light of the current understanding of central carbon metabolism in S. solfataricus and will help to obtain a system-wide understanding of this organism.
Temperature-dependent structural and functional features of a hyperthermostable enzyme using elastic neutron scattering
Koutsopoulos, S. ; Oost, J. van der; Norde, W. - \ 2005
Proteins : Structure, Function, and Bioinformatics 61 (2005)2. - ISSN 0887-3585 - p. 377 - 384.
archaeon pyrococcus-furiosus - crystal-structure - protein dynamics - 2.5-angstrom resolution - angstrom resolution - citrate synthase - thermostability - dehydrogenase - stability - fluctuations
The dynamic behavior of an endoglucanase from the hyperthermophilic microorganism Pyrococcus furiosus was investigated using elastic neutron scattering. The temperature dependence of the atomic motions was correlated with conformational and functional characteristics of the enzyme. The onset of biological function at temperatures higher than approximately 25°C (the hyperthermostable enzyme is essentially inactive at room temperature) was associated with a dynamical transition in the anharmonic motions domain. This transition from the nonactive to the enzymatically active conformation involved structurally similar conformational substates in the energy landscape. From the mean-square displacement of the protein atoms, the molecular flexibility and the effective force constants were calculated at different temperature zones. The results showed that the activity increases at higher temperatures where the intramolecular bonds are weakened and the overall rigidity of the protein is decreased. Further temperature increase resulted in significantly increased atomic fluctuations featuring heat denaturation of the protein
Ab Initio QM/MM Modeling of the Hydroxylation Step in p-Hydroxybenzoate hydroxylase
Ridder, L. ; Harvey, J.N. ; Rietjens, I.M.C.M. ; Vervoort, J.J.M. ; Mulholland, A.J. - \ 2003
The Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical 107 (2003). - ISSN 1520-6106 - p. 2118 - 2126.
potential-energy surface - pseudomonas-fluorescens - aromatic hydroxylation - absorption-spectra - phenol hydroxylase - reaction-mechanism - crystal-structure - citrate synthase - hybrid quantum - derivatives
p-Hydroxybenzoate hydroxylase (PHBH) is the model enzyme for the microbial flavin-dependent mono-oxygenases. The aromatic hydroxylation of p-hydroxybenzoate by the reactive C4a-hydroperoxyflavin cofactor intermediate in PHBH has been studied by a combined ab initio quantum mechanics and molecular mechanics (QM/MM) method. Starting from a model of the C4a-hydroperoxyflavin intermediate in the PHBH reaction cycle, built on the basis of the crystal structure of the enzyme-substrate complex, a pathway for the hydroxylation step was calculated by imposing a reaction coordinate involving cleavage of the peroxide oxygen-oxygen bond and bond formation between the C3 atom of the substrate and the distal oxygen of the peroxide moiety of the cofactor. A QM/MM potential was used in which the QM region (49 atoms) was treated at the ab initio HF level with the 3-21G(d) or 6-31G(d) basis sets. The accuracy of various aspects of the QM/MM method for this system has been tested by comparison to higher-level calculations. Inclusion of electron correlation, applied here as B3LYP/6-311+G(d,p) and LMP2/6-31+G(d) single point energy corrections to the ab initio QM/MM structures, is shown to be essential to obtain barriers in agreement with the experimental rate constant. The calculated pathways support electrophilic aromatic substitution as the mechanism of this rate-limiting step in the PHBH catalyzed reaction cycle. The polarization of the QM region by the enzyme has been investigated. A potentially important catalytic interaction between the reacting OH group in the transition state (formally OH+) and the backbone carbonyl of the Pro293 residue is identified from the calculations and is analyzed in detail. This interaction is calculated to lower the barrier by a catalytically significant 2-3 kcal/mol, corresponding to a 100-fold rate enhancement.
Quantum Mechanical/Molecular Mechanical Free Energy Simulations of the Glutathione S-Transferase (M1-1) Reaction with Phenanthrene 9,10-Oxide
Ridder, L. ; Rietjens, I.M.C.M. ; Vervoort, J.J.M. ; Mulholland, A.J. - \ 2002
Journal of the American Chemical Society 124 (2002)33. - ISSN 0002-7863 - p. 9926 - 9936.
direct dynamics calculations - active-site - catalytic mechanism - molecular-dynamics - hydrogen-bond - computer-simulation - citrate synthase - enzyme reaction - aromatic hydroxylation - reaction pathway
Glutathione S-transferases (GSTs) play an important role in the detoxification of xenobiotics in mammals. They catalyze the conjugation of glutathione to a wide range of electrophilic compounds. Phenanthrene 9,10-oxide is a model substrate for GSTs, representing an important group of epoxide substrates. In the present study, combined quantum mechanical/molecular mechanical (QM/MM) simulations of the conjugation of glutathione to phenanthrene 9,10-oxide, catalyzed by the M1-1 isoenzyme from rat, have been carried out to obtain insight into details of the reaction mechanism and the role of solvent present in the highly solvent accessible active site. Reaction-specific AM1 parameters for sulfur have been developed to obtain an accurate modeling of the reaction, and QM/MM solvent interactions in the model have been calibrated. Free energy profiles for the formation of two diastereomeric products were obtained from molecular dynamics simulations of the enzyme, using umbrella sampling and weighted histogram analysis techniques. The barriers (20 kcal/mol) are in good agreement with the overall experimental rate constant and with the formation of equal amounts of the two diastereomeric products, as experimentally observed. Along the reaction pathway, desolvation of the thiolate sulfur of glutathione is observed, in agreement with solvent isotope experiments, as well as increased solvation of the epoxide oxygen of phenanthrene 9,10-oxide, illustrating an important stabilizing role for active site solvent molecules. Important active site interactions have been identified and analyzed. The catalytic effect of Tyr115 through a direct hydrogen bond with the epoxide oxygen of the substrate, which was proposed on the basis of the crystal structure of the (9S,10S) product complex, is supported by the simulations. The indirect interaction through a mediating water molecule, observed in the crystal structure of the (9R,10R) product complex, cannot be confirmed to play a role in the conjugation step. A selection of mutations is modeled. The Asn8Asp mutation, representing one of the differences between the M1-1 and M2-2 isoenzymes, is identified as a possible factor contributing to the difference in the ratio of product formation by these two isoenzymes. The QM/MM reaction pathway simulations provide new and detailed insight into the reaction mechanism of this important class of detoxifying enzymes and illustrate the potential of QM/MM modeling to complement experimental data on enzyme reaction mechanisms