Computer-based studies on enzyme catalysis : from structure to activity

Theoretical simulations are becoming increasingly important for our understanding of how enzymes work. The aim of the research presented in this thesis is to contribute to this development by applying various computational methods to three enzymes of theβ-ketoadipate pathway, and to validate the models obtained by means of quantitative structure-activity relationships (QSAR). The models and the resulting QSARs provide valuable mechanistic information about the relevant (rate-limiting) steps in the reaction cycles of the enzymes studied.Two of the enzymes that have been studied in this thesis, are flavin dependent monooxygenases: para -hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescence , and phenol hydroxylase (PH) from Trichosporon cutaneum . These enzymes catalyse the ortho-hydroxylation of para -hydroxybenzoate and phenol, leading to the formation of catechol and protocatechuate respectively. These products are the key intermediates in the degradation of many aromatic compounds. Once the catechol or protocatechuate is formed, the aromatic ring is cleaved between the two hydroxyl-substituted carbon atoms. This intradiol cleavage is catalysed by another enzyme studied in this thesis, catechol-1,2-dioxygenase (1,2-CTD), and by protocatechuate-3,4-dioxygenase (3,4-PCD), respectively.Catechol dioxygenaseThe reaction mechanism of catechol-1,2-dioxygenase from Pseudomonas putida has been studied by means of a QSAR approach based on gas-phase molecular orbital calculations. Catechol-1,2-dioxygenase catalyses intradiol cleavage of the aromatic ring of catechol by incorporating both oxygen atoms of molecular oxygen. In addition to the native catechol, this enzyme converts several C4-substituted catechol derivatives. In this study, the 4-methyl-, 4-fluoro-, 4-chloro-, 4-bromo, 4,5-difluoro- and 4-chloro,5-fluoro-catechols were obtained biosynthetically from the corresponding phenols by using the enzyme phenol hydroxylase. The overall rate constant for their conversion by catechol-1,2-dioxygenase was determined through steady-state kinetic experiments at various oxygen concentrations and saturating catechol concentrations.The crucial step in the reaction mechanism of the enzyme catalysed reaction was considered to be the nucleophilic attack of the substrate on the oxygen molecule. Therefore, the experimental results were compared to calculated energies of the highest occupied molecular orbital (HOMO) of the various catechol substrates, which represent their nucleophilic reactivities. A (linear) correlation was found between the calculated HOMO energies and the logarithm of the experimental rate constants. This indicates that the rate-limiting step in the overall reaction cycle involves a nucleophilic reaction of the substrate. Thus, the reaction of the substrate with molecular oxygen may indeed be rate limiting. Additional calculations excluded two other steps in the reaction cycle as being rate limiting.The results for catechol-1,2-dioxygenase from Pseudomonas putida were also compared to the data from two different types of catechol-1,2-dioxygenase, a normal (type I) and a chloro-catechol dioxygenase (type II), from Pseudomonas sp. B13. It could be argued that the difference in substrate preference between both types of catechol dioxygenases is related to a differential effect of the substituents on the rate of oxygen affinity binding by the two enzymes, rather than on the rate-limiting step.p-Hydroxybenzoate hydroxylaseAn important step that has been made in this thesis is the use of a combined quantum mechanical/molecular mechanical (QM/MM) method. Using this method, the quantum mechanical (reaction pathway) calculation of the reacting compounds could be performed within the actual environment of the protein. The surrounding protein atoms are calculated at a molecular mechanical (MM) level and their electrostatic and steric effects on the quantum mechanical system are included. This QM/MM technique has been applied to the hydroxylation step catalysed by p -hydroxybenzoate hydroxylase (PHBH). It was first investigated whether the energy barriers obtained from QM/MM reaction pathway calculations could be used to explain the variation in the overall rate constants for the conversion of a series of fluorinated substrates by PHBH. Reaction pathways were calculated for the proposed rate-limiting step in the reaction cycle: the electrophilic attack of the C4a-hydroxyperoxyflavin cofactor intermediate on the substrate. The energy profiles calculated for this reaction step with the various substrates yielded barriers with different heights. A correlation was found between the natural logarithm of the experimental overall rate constants for conversion of the fluorinated substrates by PHBH and the QM/MM calculated energy barriers for the different substrates. This correlation with overall rate constants supports that the electrophilic attack of the C4a-hydroxyperoxyflavin on the substrate is indeed the rate-limiting step in the reaction cycle.The correlation also indicates that the QM/MM model provides a realistic description of the hydroxylation step, as it accounts correctly for the effect of substrate substituents on the rate of hydroxylation. This was the basis for a further and more detailed analysis of the QM/MM model, which provided detailed insight into the mechanism of substrate and cofactor activation to facilitate the electrophilic reaction. Deprotonation of the substrate, which has been observed experimentally, is shown to significantly lower the energy barrier for the calculated reaction pathway. Also, the QM/MM model allowed the analysis of the energetic effect of the individual amino acid residues on the hydroxylation reaction. The results suggest catalytic effects of a backbone carbonyl moiety (Pro293), by a specific stabilisation of the transition state, and of a (crystal) water molecule (Wat717), which stabilises the negative charge arising on the proximal oxygen of the flavin cofactor.Phenol hydroxylaseThe QM/MM technique has also been applied to phenol hydroxylase (PH). As for PHBH, the hydroxylation step, proposed to be rate limiting in the reaction cycle of PH, has been simulated for a series of halogenated substrate derivatives. The energy barriers obtained correlate well with the logarithm of the overall rate constants. This correlation supports that the electrophilic attack of the C4a-hydroperoxyflavin on the substrate is the rate-limiting step in the reaction cycle at pH 7.6 and 25°C. An additional mechanistic question addressed in this study is the protonation state of the substrate during hydroxylation. Substrate deprotonation as a mechanism of activation has not been established for PH as firmly as it has been for PHBH. Proton transfer from phenol to a potential active site base, Asp54, has been investigated by calculating a 2-dimensional potential energy surface for the two reaction coordinates, i.e. hydroxylation and proton transfer. This potential energy surface suggests that proton transfer prior to hydroxylation is the most favourable mechanism, which indicates that in the PH reaction substrate deprotonation is important as well. The QM/MM model was further analysed to provide insight into the effect of the protein environment on the simulated reaction steps. Some catalytic effects on the hydroxylation step, i.e. of a proline carbonyl moiety and of a crystal water in the active site of PH, were similar to those found for PHBH.All together, the research presented in this thesis has made a new contribution to the development and validation of computational models that can be used to address a major challenge in the present field of biochemistry, i.e. to obtain insight into enzymatic reaction mechanisms and enzyme activity on the basis of the structure of enzyme and substrate(s). Special emphasis has been on the application and validation of the QM/MM technique in the context of a QSAR approach. The investigations of this thesis provide a first survey of the possibilities of the QM/MM method with respect to the prediction of biochemical activity, taking explicitly into account the influence of the active site surroundings.</p

Computer-based studies on enzyme catalysis : from structure to activity

Theoretical simulations are becoming increasingly important for our understanding of how enzymes work. The aim of the research presented in this thesis is to contribute to this development by applying various computational methods to three enzymes of theβ-ketoadipate pathway, and to validate the models obtained by means of quantitative structure-activity relationships (QSAR). The models and the resulting QSARs provide valuable mechanistic information about the relevant (rate-limiting) steps in the reaction cycles of the enzymes studied.Two of the enzymes that have been studied in this thesis, are flavin dependent monooxygenases: para -hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescence , and phenol hydroxylase (PH) from Trichosporon cutaneum . These enzymes catalyse the ortho-hydroxylation of para -hydroxybenzoate and phenol, leading to the formation of catechol and protocatechuate respectively. These products are the key intermediates in the degradation of many aromatic compounds. Once the catechol or protocatechuate is formed, the aromatic ring is cleaved between the two hydroxyl-substituted carbon atoms. This intradiol cleavage is catalysed by another enzyme studied in this thesis, catechol-1,2-dioxygenase (1,2-CTD), and by protocatechuate-3,4-dioxygenase (3,4-PCD), respectively.Catechol dioxygenaseThe reaction mechanism of catechol-1,2-dioxygenase from Pseudomonas putida has been studied by means of a QSAR approach based on gas-phase molecular orbital calculations. Catechol-1,2-dioxygenase catalyses intradiol cleavage of the aromatic ring of catechol by incorporating both oxygen atoms of molecular oxygen. In addition to the native catechol, this enzyme converts several C4-substituted catechol derivatives. In this study, the 4-methyl-, 4-fluoro-, 4-chloro-, 4-bromo, 4,5-difluoro- and 4-chloro,5-fluoro-catechols were obtained biosynthetically from the corresponding phenols by using the enzyme phenol hydroxylase. The overall rate constant for their conversion by catechol-1,2-dioxygenase was determined through steady-state kinetic experiments at various oxygen concentrations and saturating catechol concentrations.The crucial step in the reaction mechanism of the enzyme catalysed reaction was considered to be the nucleophilic attack of the substrate on the oxygen molecule. Therefore, the experimental results were compared to calculated energies of the highest occupied molecular orbital (HOMO) of the various catechol substrates, which represent their nucleophilic reactivities. A (linear) correlation was found between the calculated HOMO energies and the logarithm of the experimental rate constants. This indicates that the rate-limiting step in the overall reaction cycle involves a nucleophilic reaction of the substrate. Thus, the reaction of the substrate with molecular oxygen may indeed be rate limiting. Additional calculations excluded two other steps in the reaction cycle as being rate limiting.The results for catechol-1,2-dioxygenase from Pseudomonas putida were also compared to the data from two different types of catechol-1,2-dioxygenase, a normal (type I) and a chloro-catechol dioxygenase (type II), from Pseudomonas sp. B13. It could be argued that the difference in substrate preference between both types of catechol dioxygenases is related to a differential effect of the substituents on the rate of oxygen affinity binding by the two enzymes, rather than on the rate-limiting step.p-Hydroxybenzoate hydroxylaseAn important step that has been made in this thesis is the use of a combined quantum mechanical/molecular mechanical (QM/MM) method. Using this method, the quantum mechanical (reaction pathway) calculation of the reacting compounds could be performed within the actual environment of the protein. The surrounding protein atoms are calculated at a molecular mechanical (MM) level and their electrostatic and steric effects on the quantum mechanical system are included. This QM/MM technique has been applied to the hydroxylation step catalysed by p -hydroxybenzoate hydroxylase (PHBH). It was first investigated whether the energy barriers obtained from QM/MM reaction pathway calculations could be used to explain the variation in the overall rate constants for the conversion of a series of fluorinated substrates by PHBH. Reaction pathways were calculated for the proposed rate-limiting step in the reaction cycle: the electrophilic attack of the C4a-hydroxyperoxyflavin cofactor intermediate on the substrate. The energy profiles calculated for this reaction step with the various substrates yielded barriers with different heights. A correlation was found between the natural logarithm of the experimental overall rate constants for conversion of the fluorinated substrates by PHBH and the QM/MM calculated energy barriers for the different substrates. This correlation with overall rate constants supports that the electrophilic attack of the C4a-hydroxyperoxyflavin on the substrate is indeed the rate-limiting step in the reaction cycle.The correlation also indicates that the QM/MM model provides a realistic description of the hydroxylation step, as it accounts correctly for the effect of substrate substituents on the rate of hydroxylation. This was the basis for a further and more detailed analysis of the QM/MM model, which provided detailed insight into the mechanism of substrate and cofactor activation to facilitate the electrophilic reaction. Deprotonation of the substrate, which has been observed experimentally, is shown to significantly lower the energy barrier for the calculated reaction pathway. Also, the QM/MM model allowed the analysis of the energetic effect of the individual amino acid residues on the hydroxylation reaction. The results suggest catalytic effects of a backbone carbonyl moiety (Pro293), by a specific stabilisation of the transition state, and of a (crystal) water molecule (Wat717), which stabilises the negative charge arising on the proximal oxygen of the flavin cofactor.Phenol hydroxylaseThe QM/MM technique has also been applied to phenol hydroxylase (PH). As for PHBH, the hydroxylation step, proposed to be rate limiting in the reaction cycle of PH, has been simulated for a series of halogenated substrate derivatives. The energy barriers obtained correlate well with the logarithm of the overall rate constants. This correlation supports that the electrophilic attack of the C4a-hydroperoxyflavin on the substrate is the rate-limiting step in the reaction cycle at pH 7.6 and 25°C. An additional mechanistic question addressed in this study is the protonation state of the substrate during hydroxylation. Substrate deprotonation as a mechanism of activation has not been established for PH as firmly as it has been for PHBH. Proton transfer from phenol to a potential active site base, Asp54, has been investigated by calculating a 2-dimensional potential energy surface for the two reaction coordinates, i.e. hydroxylation and proton transfer. This potential energy surface suggests that proton transfer prior to hydroxylation is the most favourable mechanism, which indicates that in the PH reaction substrate deprotonation is important as well. The QM/MM model was further analysed to provide insight into the effect of the protein environment on the simulated reaction steps. Some catalytic effects on the hydroxylation step, i.e. of a proline carbonyl moiety and of a crystal water in the active site of PH, were similar to those found for PHBH.All together, the research presented in this thesis has made a new contribution to the development and validation of computational models that can be used to address a major challenge in the present field of biochemistry, i.e. to obtain insight into enzymatic reaction mechanisms and enzyme activity on the basis of the structure of enzyme and substrate(s). Special emphasis has been on the application and validation of the QM/MM technique in the context of a QSAR approach. The investigations of this thesis provide a first survey of the possibilities of the QM/MM method with respect to the prediction of biochemical activity, taking explicitly into account the influence of the active site surroundings

Effects of land use changes on water and nitrogen flows at the scale of West African inland valleys: an explorative model.

Land use and cover, as influenced by agricultural practices, and the changes in these with increasing pressure on land, are among the factors determining water flows in inland valleys. Changing water flows affect nitrogen flows both at the plot level and at levels higher than plots. We present a conceptual model to show the potential influence of changes in land use on water and nitrogen flows. The model first calculates the three-dimensional redistribution of water at the inland valley level using topographic characteristics as input. For that purpose, the inland valley is divided in columns. Subsequently, the annual one-dimensional water balance is computed for each column using transfer functions. These functions use soil characteristics as input variables. Finally, the annual nitrogen balance is calculated with an equation which uses the calculated infiltration as input variable and differentiates between nitrogen uptake efficiency of annual and perennial vegetation. Both this equation and the transfer functions were tested for West Africa in an earlier study. The model-runs simulating different land-use scenarios illustrate that effects of changes in land use on water and nitrogen flows are not additive, when spatial effects of water redistribution are explicitly modelled. The addition of an explicit time dimension will make the model more dynamic in order to analyse the effects of patterns of land use throughout the year on water and nitrogen flows. The model can be used to target further research on the processes underlying water and nitrogen flows in inland valleys: in particular, the scale dependency of run-off in relation to agricultural practices merits attention in field experiments

Magnitudes of disturbance in the evolution of agro-ecosytems: N-flows in rice-based systems.

The processes underlying ecological sustainability are studied through a theoretical sequence of rice-based agro-ecosystems reflecting increasing intensities of land use. Data and assumptions are derived from existing literature in order to understand changes in the magnitude of N-flows across systems, based on N inputs equal to N outputs at the aggregated level. The three systems comprise (A) shifting cultivation, (B) mixed livestock-food crop farming, and (C) irrigated rice monoculture, that are compared on a total area basis (20, 2 and 1 ha respectively) and on a basis of per hectare crop land. Total and per hectare N inputs and outputs as well as the efficiency (ratio of N in useful outputs to N in other outputs) are estimated. On a hectare basis flows in the shifting cultivation system are less than 50 % of those in mixed livestock-cropping and only 4 % of those in rice monocropping, while efficiency increases from 0.12 to 0.29 and 0.41. Land use intensification thus leads to greater N-flows in the system, coupled with greater losses, proportionally to the increases in flows, and higher efficiency. The theoretical approach demonstrates the importance of including both magnitudes and efficiencies of N-flows at the aggregated system level in analysing ecological sustainability of agro-ecosystems

Automatic Compound Annotation from Mass Spectrometry Data Using MAGMa.

The MAGMa software for automatic annotation of mass spectrometry based fragmentation data was applied to 16 MS/MS datasets of the CASMI 2013 contest. Eight solutions were submitted in category 1 (molecular formula assignments) and twelve in category 2 (molecular structure assignment). The MS/MS peaks of each challenge were matched with in silico generated substructures of candidate molecules from PubChem, resulting in penalty scores that were used for candidate ranking. In 6 of the 12 submitted solutions in category 2, the correct chemical structure obtained the best score, whereas 3 molecules were ranked outside the top 5. All top ranked molecular formulas submitted in category 1 were correct. In addition, we present MAGMa results generated retrospectively for the remaining challenges. Successful application of the MAGMa algorithm required inclusion of the relevant candidate molecules, application of the appropriate mass tolerance and a sufficient degree of in silico fragmentation of the candidate molecules. Furthermore, the effect of the exhaustiveness of the candidate lists and limitations of substructure based scoring are discussed

Automated Annotation of Microbial and Human Flavonoid-Derived Metabolites

Flavonoids are a class of natural compounds essentially produced by plants that are part of animal and human diets and have assumed health-promoting benefits. Upon human consumption, these flavonoids are to a modest extent absorbed in the small intestines. The major part arrives in the colon where the microflora utilises and converts the flavonoids to a wide range of products. Many of these products are absorbed in the major intestines and subsequently metabolised by the host. To understand the impact of the microflora on the metabolism and possible effects on human health, complete (and quantitative) identification of the microbial as well as human metabolic conversion products of flavonoids is required. This is a challenging task, as these bioconversion products are often present in relatively small amounts, making classical identification strategies based on (accurate) mass information or nuclear magnetic resonance, not straightforward. In the absence of reference compounds, annotation of a component may be achieved by detailed expert evaluation, e.g. by searching for similar fragmentation patterns in spectral databases of known compounds. However, such manual analysis is a tedious task, and in advanced metabolite profiling experiments, with large numbers of unknown metabolites, this is a major bottleneck. Therefore, new strategies are needed for quick and reliable identification of the diverse range of molecules in complex matrices (faeces, blood, urine). Intelligent software for annotation and identification of unknowns is crucial to fully exploit complex datasets. We developed a new software tool (MAGMA) for (sub)structure-based annotation of LC-MSn datasets which, combined with a newly established database for phenolic molecules (MetIDB), enables semiautomated identification of flavonoid derivatives

Automatic Compound Annotation from Mass Spectrometry Data Using MAGMa.

The MAGMa software for automatic annotation of mass spectrometry based fragmentation data was applied to 16 MS/MS datasets of the CASMI 2013 contest. Eight solutions were submitted in category 1 (molecular formula assignments) and twelve in category 2 (molecular structure assignment). The MS/MS peaks of each challenge were matched with in silico generated substructures of candidate molecules from PubChem, resulting in penalty scores that were used for candidate ranking. In 6 of the 12 submitted solutions in category 2, the correct chemical structure obtained the best score, whereas 3 molecules were ranked outside the top 5. All top ranked molecular formulas submitted in category 1 were correct. In addition, we present MAGMa results generated retrospectively for the remaining challenges. Successful application of the MAGMa algorithm required inclusion of the relevant candidate molecules, application of the appropriate mass tolerance and a sufficient degree of in silico fragmentation of the candidate molecules. Furthermore, the effect of the exhaustiveness of the candidate lists and limitations of substructure based scoring are discussed

Magnitudes of disturbance in the evolution of agro-ecosytems: N-flows in rice-based systems.

The processes underlying ecological sustainability are studied through a theoretical sequence of rice-based agro-ecosystems reflecting increasing intensities of land use. Data and assumptions are derived from existing literature in order to understand changes in the magnitude of N-flows across systems, based on N inputs equal to N outputs at the aggregated level. The three systems comprise (A) shifting cultivation, (B) mixed livestock-food crop farming, and (C) irrigated rice monoculture, that are compared on a total area basis (20, 2 and 1 ha respectively) and on a basis of per hectare crop land. Total and per hectare N inputs and outputs as well as the efficiency (ratio of N in useful outputs to N in other outputs) are estimated. On a hectare basis flows in the shifting cultivation system are less than 50 % of those in mixed livestock-cropping and only 4 % of those in rice monocropping, while efficiency increases from 0.12 to 0.29 and 0.41. Land use intensification thus leads to greater N-flows in the system, coupled with greater losses, proportionally to the increases in flows, and higher efficiency. The theoretical approach demonstrates the importance of including both magnitudes and efficiencies of N-flows at the aggregated system level in analysing ecological sustainability of agro-ecosystems

Effects of land use changes on water and nitrogen flows at the scale of West African inland valleys: an explorative model.

Land use and cover, as influenced by agricultural practices, and the changes in these with increasing pressure on land, are among the factors determining water flows in inland valleys. Changing water flows affect nitrogen flows both at the plot level and at levels higher than plots. We present a conceptual model to show the potential influence of changes in land use on water and nitrogen flows. The model first calculates the three-dimensional redistribution of water at the inland valley level using topographic characteristics as input. For that purpose, the inland valley is divided in columns. Subsequently, the annual one-dimensional water balance is computed for each column using transfer functions. These functions use soil characteristics as input variables. Finally, the annual nitrogen balance is calculated with an equation which uses the calculated infiltration as input variable and differentiates between nitrogen uptake efficiency of annual and perennial vegetation. Both this equation and the transfer functions were tested for West Africa in an earlier study. The model-runs simulating different land-use scenarios illustrate that effects of changes in land use on water and nitrogen flows are not additive, when spatial effects of water redistribution are explicitly modelled. The addition of an explicit time dimension will make the model more dynamic in order to analyse the effects of patterns of land use throughout the year on water and nitrogen flows. The model can be used to target further research on the processes underlying water and nitrogen flows in inland valleys: in particular, the scale dependency of run-off in relation to agricultural practices merits attention in field experiments