| 1. |
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| 2. |
- Hammer, Stephan C., et al.
(författare)
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Squalene hopene cyclases : highly promiscuous and evolvable catalysts for stereoselective CC and CX bond formation
- 2013
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Ingår i: Current opinion in chemical biology. - : Elsevier. - 1367-5931 .- 1879-0402. ; 17:2, s. 293-300
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Tidskriftsartikel (refereegranskat)abstract
- A review. We review here how the inherent promiscuous nature, as well as the evolvability of terpene cyclase enzymes enables new applications in chem. We mainly focus on squalene hopene cyclases, class II triterpene synthases that use a proton-initiated cationic polycyclization cascade to form carbopolycyclic products. We highlight recent findings to demonstrate that these enzymes are capable of activating different functionalities other than the traditional terminal isoprene CC-group as well as being compatible with a wide range of nucleophiles beyond the 'ene-functionality'. Thus, squalene hopene cyclases demonstrate a great potential to be used as a toolbox for general Bronsted acid catalysis.
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| 3. |
- Hammer, Stephan C., et al.
(författare)
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Stereoselective Friedel-Crafts alkylation catalyzed by squalene hopene cyclases
- 2012
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Ingår i: Tetrahedron. - : Elsevier Ltd.. - 0040-4020 .- 1464-5416. ; 68:Copyright (C) 2013 American Chemical Society (ACS). All Rights Reserved., s. 7624-7629
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Tidskriftsartikel (refereegranskat)abstract
- In org. synthesis the Friedel-Crafts alkylation is of eminent importance, as it is a key reaction in many synthetic routes. A general access to enzymic Friedel-Crafts alkylations would be very beneficial due to the high selectivity of biocatalysts. We used designed polyprenyl Ph ethers to specifically address this reaction by using squalene hopene cyclases as catalysts. Polycyclic products with arom. rings constituting important biol. active compds. were obtained. Our results demonstrate that squalene hopene cyclases can be utilized for Friedel-Crafts alkylations and reveal the potential of these enzymes for chiral Bronsted acid catalysis.
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| 4. |
- Marton, Z., et al.
(författare)
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Mutations in the stereospecificity pocket and at the entrance of the active site of Candida antarctica lipase B enhancing enzyme enantioselectivity
- 2010
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Ingår i: Journal of Molecular Catalysis B. - : Elsevier BV. - 1381-1177 .- 1873-3158. ; 65:1-4, s. 11-17
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Tidskriftsartikel (refereegranskat)abstract
- Two different parts of Candida antarctica lipase B (stereospecificity pocket at the bottom of the active site and hydrophobic tunnel leading to the active site) were redesigned by single- or double-point mutations, in order to better control and improve enzyme enantioselectivity toward secondary alcohols. Single-point isosteric mutations of Ser47 and Thr42 situated in the stereospecificity pocket gave rise to variants with doubled enantioselectivity toward pentan-2-ol, in solid/gas reactor. Besides, the width and shape of the hydrophobic tunnel leading to the active site was modified by producing the following single-point mutants: Ile189Ala, Leu278Val and Ala282Leu. For each of these variants a significant modification of enantioselectivity was observed compared to wild-type enzyme, indicating that discrimination of the enantiomers by the enzyme could also arise from their different accessibilities from the enzyme surface to the catalytic site. (C) 2010 Elsevier B.V. All rights reserved.
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| 5. |
- Seitz, Miriam, et al.
(författare)
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Synthesis of Heterocyclic Terpenoids by Promiscuous Squalene-Hopene Cyclases
- 2013
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Ingår i: ChemBioChem. - : Wiley-VCH Verlag GmbH & Co. KGaA. - 1439-4227 .- 1439-7633. ; 14:Copyright (C) 2013 American Chemical Society (ACS). All Rights Reserved., s. 436-439
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Tidskriftsartikel (refereegranskat)abstract
- Promiscuous enzymes: The substrate promiscuity of squalene–hopene cyclases has been explored and applied in the enzyme‐catalyzed synthesis of heterocyclic terpenoids. Features of this work include cyclization reactions without pyrophosphate activation, and stereospecific ring closure of substrates of varying chain length and terminal nucleophile. This provides a biocatalytic alternative to traditional chemical catalysts.
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| 6. |
- Syrén, Per-Olof, et al.
(författare)
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Amidases have a hydrogen bond that facilitates nitrogen inversion but esterases have not
- 2011
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Ingår i: ChemCatChem. - : Wiley. - 1867-3899 .- 1867-3880. ; 3:5, s. 853-860
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Tidskriftsartikel (refereegranskat)abstract
- The fact that proteases/amidases can hydrolyze amides efficiently whereas esterases can not has been discussed during the last decades. By using molecular modeling we have found a hydrogen bond in the transition state for protease/amidase catalyzed hydrolysis of peptides and amides donated by the scissile NH-group of the substrate. The hydrogen-bond acceptor was found either in the enzyme (enzyme assisted) or in the substrate (substrate assisted). This new interaction with the NH-hydrogen in the transition state (TS) was found in sixteen proteases/amidases, which represent ten different reaction mechanisms and eleven different folding families. Esterases lack this interaction and, therefore, they are slow in hydrolyzing amides. By mimicking the substrate-assisted catalysis found in amidases we were able to shift reaction specificity of amide over ester synthesis of Candida antarctica lipase B one hundred fold. We propose that the hydrogen bond facilitates nitrogen inversion in amidases.
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| 7. |
- Syrén, Per-Olof, et al.
(författare)
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Entropy is Key to the Formation of Pentacyclic Terpenoids by Enzyme-Catalyzed Polycyclization
- 2014
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Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 53:19, s. 4845-4849
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Tidskriftsartikel (refereegranskat)abstract
- Polycyclizations constitute a cornerstone of chemistry and biology. Multicyclic scaffolds are generated by terpene cyclase enzymes in nature through a carbocationic polycyclization cascade of a prefolded polyisoprene backbone, for which electrostatic stabilization of transient carbocationic species is believed to drive catalysis. Computational studies and site-directed mutagenesis were used to assess the contribution of entropy to the polycyclization cascade catalyzed by the triterpene cyclase from A. acidocaldarius. Our results show that entropy contributes significantly to the rate enhancement through the release of water molecules through specific channels. A single rational point mutation that results in the disruption of one of these water channels decreased the entropic contribution to catalysis by 60kcalmol(-1). This work demonstrates that entropy is the key to enzyme-catalyzed polycyclizations, which are highly relevant in biology since 90% of all natural products contain a cyclic subunit.
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| 8. |
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| 9. |
- Syren, Per-Olof, et al.
(författare)
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Increased activity of enzymatic transacylation of acrylates through rational design of lipases
- 2010
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Ingår i: Journal of Molecular Catalysis B. - : Elsevier BV. - 1381-1177 .- 1873-3158. ; 65:1-4, s. 3-10
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Tidskriftsartikel (refereegranskat)abstract
- A rational design approach was used to create the mutant Candida antarctica lipase B (CALB, also known as Pseudozyma antarctica lipase B) V190A having a k(cat) three times higher compared to that of the wild type (wt) enzyme for the transacylation of the industrially important compound methyl methacrylate. The enzymatic contribution to the transacylation of various acrylates and corresponding saturated esters was evaluated by comparing the reaction catalysed by CALB wt with the acid (H2SO4) catalysed reaction. The performances of CALB wt and mutants were compared to two other hydrolases, Humicola insolens cutinase and Rhizomucor mihei lipase. The low reaction rates of enzyme catalysed transacylation of acrylates were found to be caused mainly by electronic effects due to the double bond present in this class of molecules. The reduction in rate of enzyme catalysed transacylation of acrylates compared to that of the saturated ester methyl propionate was however less than what could be predicted from the energetic cost of breaking the pi-system of acrylates solely. The nature and concentration of the acyl acceptor was found to have a profound effect on the reaction rate. (C) 2009 Elsevier B.V. All rights reserved.
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| 10. |
- Syrén, Per-Olof, et al.
(författare)
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Least-motion mechanism in enzyme catalysis
- 2010
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Ingår i: The FEBS Journal. - : Wiley-Blackwell Publishing Inc.. - 1742-464X .- 1742-4658. ; 277, s. 263-263
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 11. |
- Syrén, Per-Olof, 1981-
(författare)
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On electrostatic effects, minimal motion and other catalytic strategies used by enzymes
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Enzymes are powerful biocatalysts that provide rate accelerations of up to 1019 fold compared to the corresponding uncatalyzed reaction in solution. The origin of the remarkable performance displayed by enzymes has fascinated and puzzled researchers for over a hundred years. It is clear that the catalytic effect is a consequence of the higher degree of transition state stabilization for the enzyme catalyzed reaction compared to the corresponding uncatalyzed reaction. It is still not well understood exactly how this transition state stabilization occurs and the relative importance of various catalytic effects are discussed. Catalytic effects involving electrostatics, near attack conformers, dynamic effects and an economy in atomic motion are discussed in this thesis. The importance of electrostatic effects is corroborated in this thesis. A single hydrogen bond in transition state constitutes an important difference between amidases and esterases. A hydrogen bond in transition state is found in all sixteen analyzed amidases representing ten different reaction mechanisms and eleven different folding families. The hydrogen bond is shown to be either substrate assisted or enzyme assisted. The role of this hydrogen bond is to assist nitrogen inversion in amidases. Esterases lack this interaction in transition state and therefore they are very poor catalysts in the hydrolysis of amides. Electrostatic interactions are found to facilitate proton transfer that enhances the rate of lipase catalyzed N-acylation of amino alcohols. In this thesis electrostatic effects in the substrate are shown to be important for the lipase catalyzed transacylation of acrylates The α,β-double bond present in acrylates introduce electronic effects that has the consequence of restricting the conformational freedom of the substrate in its ground state to two flat conformations, s-cis and s-trans. It is shown that acrylates form near attack conformers (NACs) from their ground state s-cis/s-trans planar conformations. The ability of the enzyme to accommodate such apparent s-cis/s-trans substrate conformations dictates the probability to form productive transition states and thus the reaction rate. Dynamic effects are important in enzymes. In this thesis it is found that a point mutation increases the flexibility of a neighbouring residue in Candida antarctica lipase B. This allows the mutated enzyme to explore conformations not accessible for the wild-type enzyme. The dynamics has the effect to decrease steric interactions in transition state with concomitant rate increase for the transacylation of methyl methacrylate. In this thesis an economy of atomic motion during enzyme catalysis is observed. Nitrogen inversion in amidases constitutes an interesting example. A rotation as part of the reaction mechanism for amide bond hydrolysis would involve much more motion.
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| 12. |
- Syrén, Per-Olof, et al.
(författare)
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Proton Shuttle Mechanism in the Transition State of Lipase-Catalyzed N-Acylation of Amino Alcohols
- 2013
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Ingår i: ChemCatChem. - : Wiley-VCH Verlagsgesellschaft. - 1867-3880 .- 1867-3899. ; 5:7, s. 1842-1853
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Tidskriftsartikel (refereegranskat)abstract
- An increased reaction rate for lipase-catalyzed N-acylation of amino alcohols relative to that of monofunctionalized amines can be explained by a hydrogen shuttling mechanism that avoids nitrogen inversion in the transition state. The mechanism does not involve acyl migration from an ester intermediate that would be formed first, an explanation that permeates the literature. Our suggested reaction mechanism is dependent on the preference of amino alcohols to form intramolecular hydrogen bonds and the capability of the enzyme to accommodate and exploit the specific hydrogen bonding pattern provided by the ligand during catalysis. Our proposed proton shuttle mechanism involves the transfer of two protons in the transition state concomitant with a nucleophilic attack on the acyl enzyme and provides an explanation for the high reaction rate and chemoselectivity for lipase-catalyzed N-acylation of amino alcohols. Moreover, the proton shuttle mechanism explains the increased reaction rate for the enzyme-catalyzed N-acylation of diamines and of methoxy-2-propylamine, for which O- to N-acyl migration is impossible. A linear free-energy relationship analysis based on the experimental results showed that all of our investigated difunctionalized amine substrates afforded a substrate-assisted rate acceleration of the N-acylation by the same reaction mechanism. Furthermore, the results of the analysis were consistent with partial proton transfer in the rate-limiting transition state, which further supports our suggested proton shuttle mechanism.
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| 13. |
- Syrén, Per-Olof, et al.
(författare)
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Substrate Conformations Set the Rate of Enzymatic Acrylation by Lipases
- 2010
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 11:6, s. 802-810
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Tidskriftsartikel (refereegranskat)abstract
- Acrylates represent a class of 4-unsaturated compounds of high industrial importance. We investigated the influence of substrate conformations on the experimentally determined reaction rates of the enzyme-catalysed transacylation of methyl acrylate and derivatives by ab initio DFT B3LYP calculations and molecular dynamics simulations. The results supported a least-motion mechanism upon the sp(2) to sp(3) substrate transition to reach the transition state in the enzyme active site. This was in accordance with our hypothesis that acrylates form productive transition states from their low-energy s-sis/s-trans conformations. Apparent k(cat) values were measured for Candida antarctica lipase B (CALB), Humicola insolens cutinase and Rhizomucor miehei lipase and were compared to results from computer simulations. More potent enzymes for acryltransfer, such as the CALB mutant V190A and acrylates with higher turnover numbers, showed elevated populations of productive transition states.
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| 14. |
- Syren, Per-Olof
(författare)
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The solution of nitrogen inversion in amidases
- 2013
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Ingår i: The FEBS Journal. - : Wiley-Blackwell. - 1742-464X .- 1742-4658. ; 280:13, s. 3069-3083
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Tidskriftsartikel (refereegranskat)abstract
- An important mechanistic aspect of enzyme-catalyzed amide bond hydrolysis is the specific orientation of the lone pair of the N atom of the scissile amide bond during catalysis. As discussed in the literature during the last decades, stereoelectronic effects cause the single lone pair in the formed tetrahedral intermediate to be situated in a non-productive conformation in the enzyme active site and hence N atom inversion or rotation is necessary. By discussing recent mechanistic findings in the literature relevant for the conformation of the lone pair of the reacting amide N atom, it is demonstrated that Nature has evolved at least 2 catalytic strategies to cope with the stereoelectronic constraints inherent to amide bond hydrolysis regardless of the fold or catalytic mechanism. One soln. to the inversion problem is to stabilize the transition state of inversion by H-bond formation; another is to introduce a concerted proton shuttle mechanism that avoids inversion and delivers a hydrogen to the lone pair. Here, by using mol. modeling it was demonstrated that the H-bond strategy is general and can be expanded to include many amidases/proteases with important metabolic functions, including the proteasome. Some examples of the proton shuttle mechanism are also mentioned. To complete the picture of efficient enzyme-catalyzed amide bond hydrolysis, general interactions in the active site of these catalysts were discussed. An expanded knowledge of the prerequisites of efficient amide bond hydrolysis beyond the oxyanion hole and the catalytic dyad/triad will be of importance for enzyme and drug design. [on SciFinder(R)]
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| 15. |
- Vallin, Michaela, et al.
(författare)
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Mutant Lipase-Catalyzed Kinetic Resolution of Bulky Phenyl Alkyl sec-Alcohols : A Thermodynamic Analysis of Enantioselectivity
- 2010
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 11:3, s. 411-416
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Tidskriftsartikel (refereegranskat)abstract
- The size of the stereoselectivity pocket of Candida antarctica lipase B limits the range of alcohols that can be resolved with this enzyme. These steric constrains have been changed by increasing the size of the pocket by the mutation W104A. The mutated enzyme has good activity and enantioselectivity toward bulky secondary alcohols, such as 1-phenylalkanols, with alkyl chains up to eight carbon atoms. The S enantiomer was preferred in contrast to the wild-type enzyme, which has R selectivity. The magnitude of the enantioselectivity changes in an interesting way with the chain length of the alkyl moiety. It is governed by interplay between entropic and enthalpic contributions and substrates with long alkyl chains are resolved best with E values higher than 100. The enantioselectivity increases with temperature for the small substrates, but decreases for the long ones.
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