| 61. |
- Larsson, Anna-Karin, 1971-
(författare)
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Directed Enzyme Evolution of Theta Class Glutathione Transferase : Studies of Recombinant Libraries and Enhancement of Activity toward the Anticancer Drug 1,3-bis(2-Chloroethyl)-1-nitrosourea
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glutathione transferases (GSTs) are detoxication enzymes involved in the cellular protection against a wide range of reactive substances. The role of GSTs is to catalyze the conjugation of glutathione with electrophilic compounds, which generally results in less toxic products. The ability to catalyze the denitrosation of the anticancer drug 1,3-bis(2-chloroethyl)- 1-nitrosourea (BCNU) was measured in twelve different GSTs. Only three of the enzymes showed any measurable activity with BCNU, of which human GST T1-1 was the most efficient. This is of special interest, since human GST T1-1 is a polymorphic protein and its expression in different patients may be crucial for the response to BCNU.DNA shuffling was used to create a mutant library by recombination of cDNA coding for two different Theta-class GSTs. In total, 94 randomly picked mutants were characterized with respect to their catalytic activity with six different substrates, expression level and sequence. A clone with only one point mutation compared to wild-type rat GST T2-2 had a significantly different substrate-activity pattern. A high expressing mutant of human GST T1-1 was also identified, which is important, since the yield of the wild-type GST T1-1 is generally low. Characterization of the Theta library demonstrated divergence of GST variants both in structure and function. The properties of every mutant were treated as a point in a six-dimensional substrate-activity space. Groups of mutants were formed based on euclidian distances and K-means cluster analyses. Both methods resulted in a set of five mutants with high alkyltransferase activities toward dichloromethane and 4-nitrophenethyl bromide (NPB). The five selected mutants were used as parental genes in a new DNA shuffling. Addition of cDNA coding for mouse and rat GST T1-1 improved the genetic diversity of the library. The evolution of GST variants was directed towards increased alkyltransferase activity including activity with the anticancer drug BCNU. NPB was used as a surrogate substrate in order to facilitate the screening process. A mutant from the second generation displayed a 65-fold increased catalytic activity with NPB as substrate compared to wild-type human GST T1-1. The BCNU activity with the same mutant had increased 175-fold, suggesting that NPB is a suitable model substrate for the anticancer drug. Further evolution presented a mutant in the fifth generation of the library with 110 times higher NPB activity than wild-type human GST T1-1.
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| 62. |
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| 63. |
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| 64. |
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| 65. |
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| 66. |
- Mannervik, Bengt, 1943-
(författare)
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Molecular enzymology of the glyoxalase system.
- 2008
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Ingår i: Drug metabolism and drug interactions. - 0792-5077. ; 23:1-2, s. 13-27
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Tidskriftsartikel (refereegranskat)abstract
- The glyoxalase system catalyzes the conversion of 2-oxoaldehydes into the corresponding 2-hydroxyacids. This biotransformation involves two separate enzymes, glyoxalase I and glyoxalase II, which bring about two consecutive reactions involving the thiol-containing tripeptide glutathione as a cofactor. The physiologically most important substrate methylglyoxal is converted by glyoxalase I into S-D-lactoyl-glutathione in the first reaction. Subsequently, glyoxalase II catalyzes the hydrolysis of this thiolester into D-lactic acid and free glutathione. The structures of both enzymes have been obtained via molecular cloning, heterologous expression, and X-ray diffraction analysis. Glyoxalase I and glyoxalase II are metalloenzymes and zinc plays an essential role in their diverse catalytic mechanisms. Both enzymes appear linked to a variety of pathological conditions, but further investigations are required to clarify the different physiological aspects of the glyoxalase system.
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| 67. |
- Mannervik, Bengt, et al.
(författare)
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Multi-substrate-activity space and quasi-species in enzyme evolution : Ohno's dilemma, promiscuity and functional orthogonality
- 2009
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Ingår i: Biochemical Society Transactions. - 0300-5127 .- 1470-8752. ; 37:Pt 4, s. 740-744
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Tidskriftsartikel (refereegranskat)abstract
- A functional enzyme displays activity with at least one substrate and can be represented by a vector in substrate-activity space. Many enzymes, including GSTs (glutathione transferases), are promiscuous in the sense that they act on alternative substrates, and the corresponding vectors operate in multidimensional space. The direction of the vector is governed by the relative activities of the diverse substrates. Stochastic mutations of already existing enzymes generate populations of variants, and clusters of functionally similar mutants can serve as parents for subsequent generations of enzymes. The proper evolving unit is a functional quasi-species, which may not be identical with the 'best' variant in its generation. The manifestation of the quasi-species is dependent on the substrate matrix used to explore catalytic activities. Multivariate analysis is an approach to identifying quasi-species and to investigate evolutionary trajectories in the directed evolution of enzymes for novel functions.
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| 68. |
- Mannervik, Bengt, et al.
(författare)
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Nomenclature for mammalian soluble glutathione transferases
- 2005
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Ingår i: Methods of Enzymology. ; 401, s. 1-8
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Tidskriftsartikel (refereegranskat)abstract
- The nomenclature for human soluble glutathione transferases (GSTs) is extended to include new members of the GST superfamily that have been discovered, sequenced, and shown to be expressed. The GST nomenclature is based on primary structure similarities and the division of GSTs into classes of more closely related sequences. The classes are designated by the names of the Greek letters: Alpha, Mu, Pi, etc., abbreviated in Roman capitals: A, M, P, and so on. (The Greek characters should not be used.) Class members are distinguished by Arabic numerals and the native dimeric protein structures are named according to their subunit composition (e.g., GST A1-2 is the enzyme composed of subunits 1 and 2 in the Alpha class). Soluble GSTs from other mammalian species can be classified in the same manner as the human enzymes, and this chapter presents the application of the nomenclature to the rat and mouse GSTs.
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| 69. |
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| 70. |
- Mannervik, Bengt
(författare)
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Optimizing the heterologous expression of glutathione transferase
- 2005
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Ingår i: Methods of Enzymology. ; 401, s. 254-265
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Tidskriftsartikel (refereegranskat)abstract
- The heterologous expression of a protein may be enhanced by silent mutations in the coding region of its corresponding DNA. This simple approach has been successfully used for optimized production of a number of glutathione-linked enzymes. For example, the yield of human glutathione transferase M2-2 was elevated by 140-fold in a clone isolated by immunoscreening of a library of plasmids with randomized synonymous codons in the 5'-segment of the region encoding the enzyme.
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