| 1. |
- Amrein, Beat A., et al.
(author)
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Expanding the catalytic triad in epoxide hydrolases and related enzymes
- 2015
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In: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 5:10, s. 5702-5713
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Journal article (peer-reviewed)abstract
- Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.
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| 2. |
- Naworyta, Agata
(author)
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Structure-function studies of epoxide hydrolases
- 2010
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Doctoral thesis (other academic/artistic)abstract
- Epoxides are three-membered cyclic ethers formed in cells via several metabolic pathways. Epoxide hydrolases (EHs) are enzymes that hydrolyse epoxides to the corresponding diols. The main goal of this thesis was to investigate the structures of EHs from the alpha/beta-hydrolase family. The first part concerns the structural and functional analysis of a protein-water channel found in EHs in many plants. Thermostability studies, sequence analysis and determination of the x-ray structure of a mutated EH enzyme from Solanum tuberosum led to the conclusions that the water channel in plants participates in stabilization of the protein structure and furthermore, it forms an efficient system to enable transfer of protons that are required for enzymatic catalysis. The second part describes how computational methods together with structural and kinetic information identified factors that are responsible for the enhanced enantioselectivity of an improved variant of EH from Aspergillus niger obtained during a directed evolution process. The x-ray structure of the mutant showed that dramatic changes in the active site explain why the preferred (S)-substrate binds more easily in the active site than the disfavored (R)-enantiomer. The study underscores the importance of obtaining structural data when attempting to understand the results of directed evolution. The last part presents the structures of two novel microbial EHs that have been shown to produce chemically valuable 1,2-diols and exhibit high enantioselectivity. Their similarity to the mammalian microsomal EH, a key enzyme in detoxification, provided new information about its possible structure. The improved sequence alignment based on the structural work gives new insights on the connections between sequences/structures and the broad scope of selectivities among EHs
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| 3. |
- Reetz, Manfred T., et al.
(author)
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Directed Evolution of an Enantioselective Epoxide Hydrolase : Uncovering the Source of Enantioselectivity at Each Evolutionary Stage
- 2009
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In: Journal of the American Chemical Society. - : ACS. - 0002-7863 .- 1520-5126. ; 131:21, s. 7334-7343
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Research review (peer-reviewed)abstract
- Directed evolution of enzymes as enantioselective catalysts in organic chemistry is an alternative to traditional asymmetric catalysis using chiral transition-metal complexes or organocatalysts, the different approaches often being complementary. Moreover, directed evolution studies allow us to learn more about how enzymes perform mechanistically. The present study concerns a previously evolved highly enantioselective mutant of the epoxide hydrolase from Aspergillus niger in the hydrolytic kinetic resolution of racemic glycidyl phenyl ether. Kinetic data, molecular dynamics calculations, molecular modeling, inhibition experiments, and X-ray structural work for the wild-type (WT) enzyme and the best mutant reveal the basis of the large increase in enantioselectivity (E = 4.6 versus E = 115). The overall structures of the WT and the mutant are essentially identical, but dramatic differences are observed in the active site as revealed by the X-ray structures. All of the experimental and computational results support a model in which productive positioning of the preferred (S)-glycidyl phenyl ether, but not the (R)-enantiomer, forms the basis of enhanced enantioselectivity. Predictions regarding substrate scope and enantioselectivity of the best mutant are shown to be possible.
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| 4. |
- Russo, Francesco, et al.
(author)
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Optimization and Evaluation of 5-Styryl-Oxathiazol-2-one Mycobacterium tuberculosis Proteasome Inhibitors as Potential Antitubercular Agents
- 2015
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In: ChemistryOpen. - : Wiley. - 2191-1363. ; 4:3, s. 342-362
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Journal article (peer-reviewed)abstract
- This is the first report of 5-styryl-oxathiazol-2-ones as inhibitors of the Mycobacterium tuberculosis (Mtb) proteasome. As part of the study, the structure-activity relationship of oxathiazolones as Mtb proteasome inhibitors has been investigated. Furthermore, the prepared compounds displayed a good selectivity profile for Mtb compared to the human proteasome. The 5-styryl-oxathiazol-2-one inhibitors identified showed little activity against replicating Mtb, but were rapidly bactericidal against nonreplicating bacteria. (E)-5-(4-Chlorostyryl)-1,3,4-oxathiazol-2-one) was most effective, reducing the colony-forming units (CFU)/mL below the detection limit in only seven days at all concentrations tested. The results suggest that this new class of Mtb proteasome inhibitors has the potential to be further developed into novel antitubercular agents for synergistic combination therapies with existing drugs.
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| 5. |
- Stern, Ana Laura, et al.
(author)
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Structures of type B ribose 5-phosphate isomerase from Trypanosoma cruzi shed light on the determinants of sugar specificity in the structural family
- 2011
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In: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 278:5, s. 793-808
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Journal article (peer-reviewed)abstract
- Ribose-5-phosphate isomerase (Rpi; EC 5.3.1.6) is a key activity of the pentose phosphate pathway. Two unrelated types of sequence/structure possess this activity: type A Rpi (present in most organisms) and type B Rpi (RpiB) (in some bacteria and parasitic protozoa). In the present study, we report enzyme kinetics and crystallographic studies of the RpiB from the human pathogen, Trypanosoma cruzi. Structures of the wild-type and a Cys69Ala mutant enzyme, alone or bound to phosphate, D-ribose 5-phosphate, or the inhibitors 4-phospho-D-erythronohydroxamic acid and D-allose 6-phosphate, highlight features of the active site, and show that small conformational changes are linked to binding. Kinetic studies confirm that, similar to the RpiB from Mycobacterium tuberculosis, the T. cruzi enzyme can isomerize D-ribose 5-phosphate effectively, but not the 6-carbon sugar D-allose 6-phosphate; instead, this sugar acts as an inhibitor of both enzymes. The behaviour is distinct from that of the more closely related (to T. cruzi RpiB) Escherichia coli enzyme, which can isomerize both types of sugars. The hypothesis that differences in a phosphate-binding loop near the active site were linked to the differences in specificity was tested by construction of a mutant T. cruzi enzyme with a sequence in this loop more similar to that of E. coli RpiB; this mutant enzyme gained the ability to act on the 6-carbon sugar. The combined information allows us to distinguish the two types of specificity patterns in other available sequences. The results obtained in the present study provide insights into the action of RpiB enzymes generally, and also comprise a firm basis for future work in drug design.
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| 6. |
- Thomaeus, Ann, et al.
(author)
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Removal of distal protein-water hydrogen bonds in a plant epoxide hydrolase increases catalytic turnover but decreases thermostability
- 2008
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In: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 17:7, s. 1275-1284
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Journal article (peer-reviewed)abstract
- A putative proton wire in potato soluble epoxide hydrolase 1, StEH1, was identified and investigated by means of site-directed mutagenesis, steady-state kinetic measurements, temperature inactivation studies, and X-ray crystallography. The chain of hydrogen bonds includes five water molecules coordinated through backbone carbonyl oxygens of Pro186, Leu266, His269, and the His153 imidazole. The hydroxyl of Tyr149 is also an integrated component of the chain, which leads to the hydroxyl of Tyr154. Available data suggest that Tyr154 functions as a final proton donor to the anionic alkylenzyme intermediate formed during catalysis. To investigate the role of the putative proton wire, mutants Y149F, H153F, and Y149F/H153F were constructed and purified. The structure of the Y149F mutant was solved by molecular replacement and refined to 2.0 Å resolution. Comparison with the structure of wild-type StEH1 revealed only subtle structural differences. The hydroxyl group lost as a result of the mutation was replaced by a water molecule, thus maintaining a functioning hydrogen bond network in the proton wire. All mutants showed decreased catalytic efficiencies with the R,R-enantiomer of trans-stilbene oxide, whereas with the S,S-enantiomer, k cat/K M was similar or slightly increased compared with the wild-type reactions. k cat for the Y149F mutant with either TSO enantiomer was increased; thus the lowered enzyme efficiencies were due to increases in K M. Thermal inactivation studies revealed that the mutated enzymes were more sensitive to elevated temperatures than the wild-type enzyme. Hence, structural alterations affecting the hydrogen bond chain caused increases in k cat but lowered thermostability.
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