| 51. |
- Fedulova, Natalia, 1980-, et al.
(författare)
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Experimental Conditions Affecting Functional Comparison of Highly Active Glutathione Transferases
- 2011
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 413:1, s. 16-23
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Tidskriftsartikel (refereegranskat)abstract
- Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. Direct evidence of underestimation of activity of human GST A3-3 and porcine GST A2-2 measured at submicromolar enzyme concentrations is reported here for the first time. Combination of time-dependent and enzyme concentration-dependent loss of activity as well as the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurementsof GSTs. These effects contribute to high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Adsorption of GSTs to surfaces was found to be the main explanation of the observed phenomena. Several approaches to improved functional comparison of highly active GSTs are proposed.
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| 52. |
- Fedulova, Natalia, 1980-, et al.
(författare)
-
Porcine glutathione transferase Alpha 2-2 is a human GST A3-3 analogue that catalyses steroid double-bond isomerization
- 2010
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 431:1, s. 159-167
-
Tidskriftsartikel (refereegranskat)abstract
- A primary role of GSTs (glutathione transferases) is detoxication of electrophilic compounds. In addition to this protective function, hGST (human GST) A3-3, a member of the Alpha class of soluble GSTs, has prominent steroid double-bond isomerase activity. The isomerase reaction is an obligatory step in the biosynthesis of steroid hormones, indicating a special role of hGST A3-3 in steroidogenic tissues. An analogous GST with high steroid isomerase activity has so far not been found in any other biological species. In the present study, we characterized a Sus scrofa (pig) enzyme, pGST A2-2, displaying high steroid isomerase activity. High levels of pGST A2-2 expression were found in ovary, testis and liver. In its functional properties, other than steroid isomerization, pGST A2-2 was most similar to hGST A3-3. The properties of the novel porcine enzyme lend support to the notion that particular GSTs play an important role in steroidogenesis.
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| 53. |
- Govindarajan, Sridhar, et al.
(författare)
-
Mapping of Amino Acid Substitutions Conferring Herbicide Resistance in Wheat Glutathione Transferase
- 2015
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 4:3, s. 221-227
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Tidskriftsartikel (refereegranskat)abstract
- We have used design of experiments (DOE) and systematic variance to efficiently explore glutathione transferase substrate specificities caused by amino acid substitutions. Amino acid substitutions selected using phylogenetic analysis were synthetically combined using a DOE design to create an information-rich set of gene variants, termed infologs. We used machine learning to identify and quantify protein sequence-function relationships against 14 different substrates The resulting models were quantitative and predictive, serving as a guide for engineering of glutathione transferase activity toward a diverse set of herbicides Predictive quantitative models like those presented here have broad applicability for bioengineering.
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| 54. |
- Grahn, Elin, et al.
(författare)
-
New crystal structures of human glutathione transferase A1-1 shed light on glutathione binding and the conformation of the C-terminal helix.
- 2006
-
Ingår i: Acta Crystallogr D Biol Crystallogr. - 0907-4449. ; 62:Pt 2, s. 197-207
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Tidskriftsartikel (refereegranskat)abstract
- Human glutathione transferase A1-1 is a well studied enzyme, but despite a wealth of structural and biochemical data a number of aspects of its catalytic function are still poorly understood. Here, five new crystal structures of this enzyme are described that provide several insights. Firstly, the structure of a complex of the wild-type human enzyme with glutathione was determined for the first time at 2.0 angstroms resolution. This reveals that glutathione binds in the G site in a very similar fashion as the glutathione portion of substrate analogues in other structures and also that glutathione binding alone is sufficient to stabilize the C-terminal helix of the protein. Secondly, we have studied the complex with a decarboxylated glutathione conjugate that is known to dramatically decrease the activity of the enzyme. The T68E mutant of human glutathione transferase A1-1 recovers some of the activity that is lost with the decarboxylated glutathione, but our structures of this mutant show that none of the earlier explanations of this phenomenon are likely to be correct. Thirdly, and serendipitously, the apo structures also reveal the conformation of the crucial C-terminal region that is disordered in all previous apo structures. The C-terminal region can adopt an ordered helix-like structure even in the apo state, but shows a strong tendency to unwind. Different conformations of the C-terminal regions were observed in the apo states of the two monomers, which suggests that cooperativity could play a role in the activity of the enzyme.
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| 55. |
- Gurell, Ann, 1981-
(författare)
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Biochemical Studies on a Plant Epoxide Hydrolase : Discovery of a Proton Entry and Exit Pathway and the Use of In vitro Evolution to Shift Enantioselectivity
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The work leading to this thesis has provided additional information and novel knowledge concerning structure-function relationship in the potato epoxide hydrolase. Epoxide hydrolases are enzymes catalyzing the hydrolysis of epoxides to yield the corresponding vicinal diols. The reaction mechanism proceeds via a nucleophilic attack resulting in a covalent alkylenzyme intermediate, which in turn is attacked by a base-activated water molecule, followed by product release. Epoxides and diols are precursors in the production of chiral compounds and the use of epoxide hydrolases as biocatalysts is growing. The promising biocatalyst StEH1, a plant epoxide hydrolase from potato, has been investigated in this thesis. In paper I the active site residue Glu35, was established to be important for the formation of the alkylenzyme intermediate, activating the nucleophile for attack by facilitated proton release through a hydrogen bond network. Glu35 is also important during the hydrolytic half reaction by optimally orienting the hydrolytic water molecule, aiding in the important dual function of the histidine base. Glu35 makes it possible for the histidine to work as both an acid and a base. In paper II a putative proton wire composed of five water molecules lining a protein tunnel was proposed to facilitate effective proton transfer from the exterior to the active site, aiding in protonation of the alkylenzyme intermediate. The protein tunnel is also proposed to stabilize plant epoxide hydrolases via hydrogen bonds between water molecules and protein. Enzyme variants with modified enantiospecificity for the substrate (2,3-epoxypropyl)benzene have been constructed by in vitro evolution using the CASTing approach. Residues lining the active site pocket were targeted for mutagenesis. From the second generation libraries a quadruple enzyme variant, W106L/L109Y/V141K/I155V, displayed a radical shift in enantioselectivity. The wild-type enzyme favored the S-enantiomer with a ratio of 2:1, whereas the quadruple variant showed a 15:1 preference for the R-enantiomer.
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| 56. |
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| 57. |
- Gustafsson, Ann, et al.
(författare)
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Role of the glutamyl alpha-carboxylate of the substrate glutathione in the catalytic mechanism of human glutathione transferase A1-1
- 2001
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 40:51, s. 15835-15845
-
Tidskriftsartikel (refereegranskat)abstract
- The Glu alpha-carboxylate of glutathione contributes to the catalytic function of the glutathione transferases. The catalytic efficiency of human glutathione transferase A1-1 (GST A1-1) in the conjugation reaction with 1-chloro-2,4-dinitrobenzene is reduced 15 000-fold if the decarboxylated analogue of glutathione, dGSH (GABA-Cys-Gly), is used as an alternative thiol substrate. The decrease is partially due to an inability of the enzyme to promote ionization of dGSH. The pK(a) value of the thiol group of the natural substrate glutathione decreases from 9.2 to 6.7 upon binding to GST A1-1. However, the lack of the Glu alpha-carboxylate in dGSH raised the pK(a) value of the thiol in the enzymatic reaction to that of the nonenzymatic reaction. Furthermore, K(M)(dGSH) was 100-fold higher than K(M)(GSH). The active-site residue Thr68 forms a hydrogen bond to the Glu alpha-carboxylate of glutathione. Introduction of a carboxylate into GST A1-1 by a T68E mutation increased the catalytic efficiency with dGSH 10-fold and reduced the pK(a) value of the active site bound dGSH by approximately 1 pH unit. The altered pK(a) value is consistent with a catalytic mechanism where the carboxylate contributes to ionization of the glutathione thiol group. With Delta(5)-androstene-3,17-dione as substrate the efficiency of the enzyme is decreased 24 000-fold while with 4-nitrocinnamaldehyde (NCA) the decrease is less than 150-fold. In the latter reaction NCA accepts a proton and, unlike the other reactions studied, may not be dependent on the Glu alpha-carboxylate for deprotonation of the thiol group. An additional function of the Glu alpha-carboxylate may be productive orientation of glutathione within the active site.
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| 58. |
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| 59. |
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| 60. |
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