Screening for recombinant glutathione transferases active with monochlorobimane

Eklund, Birgitta I.; Edalat, Maryam; Stenberg, Gun; Mannervik, Bengt

doi:10.1016/s0003-2697(02)00258-0

Cited by 23 publications

(16 citation statements)

References 14 publications

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“…Many mechanisms are involved in MDR, and these include alterations in drug transport resulting in impaired entry or enhanced efflux of the drug from the tumor cell, enhanced DNA repair, alterations in target proteins, and alterations in drug metabolism 7 . The glutathione transferases (EC 2.5.1.18: GST) are a unique family of detoxification enzymes comprising a large group of cytosolic, mitochondrial, and microsomal proteins which are capable of multiple reactions with a multitude of substrates, both endogenous and xenobiotic 8 . These enzymes can constitute up to 10% of cytosolic protein in some mammalian organs and play an important role in the detoxification of electrophilic xenobiotics such as, drugs, toxins, and carcinogens allowing the products to be exported from the cell through the GS-X pump in an ATP-dependent manner 9 .…”

Section: Introductionmentioning

confidence: 99%

The evaluation of novel natural products as inhibitors of human glutathione transferase P1-1

Mukanganyama

Bezabih

Metuno

et al. 2010

Journal of Enzyme Inhibition and Medicinal Chemistry

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Section: Introductionmentioning

confidence: 99%

The evaluation of novel natural products as inhibitors of human glutathione transferase P1-1

Mukanganyama

Bezabih

Metuno

et al. 2010

Journal of Enzyme Inhibition and Medicinal Chemistry

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“…3,[4][5][6][7][8] Recently, fluorescent single-cell assays for GST activity have been developed, opening the way for screening large mutant libraries by flow cytometry. 9, 10 Griswold et al reported the construction of large libraries by homology-independent recombination of the human theta class 1-1 (hGSTT1-1) and rat theta class 2-2 (rGSTT2-2) GSTs. 11 The two parental enzymes exhibit drastically different substrate specificities, with rGSTT2-2 having >400-fold higher catalytic efficiency for the conjugation of 7-amino-4-chloromethyl coumarin (CMAC) compared to hGSTT1-1.…”

Section: Introductionmentioning

confidence: 99%

The Evolution of Catalytic Efficiency and Substrate Promiscuity in Human Theta Class 1-1 Glutathione Transferase

Griswold

Aiyappan

Iverson

et al. 2006

Journal of Molecular Biology

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Theta class glutathione transferases (GST) from various species exhibit markedly different catalytic activities in conjugating the tripeptide glutathione (GSH) to a variety of electrophilic substrates. For example, the human theta 1-1 enzyme (hGSTT1-1) is 440-fold less efficient than the rat theta 2-2 enzyme (rGSTT2-2) with the fluorogenic substrate 7-amino-4-chloromethyl coumarin (CMAC). Large libraries of hGSTT1-1 constructed by error-prone PCR, DNA shuffling, or saturation mutagenesis were screened for improved catalytic activity towards CMAC in a quantitative fashion using flow cytometry. An iterative directed evolution approach employing random mutagenesis in conjunction with homologous recombination gave rise to enzymes exhibiting up to a 20,000-fold increase in k(cat)/K(M) compared to hGSTT1-1. All highly active clones encoded one or more mutations at residues 32, 176, or 234. Combinatorial saturation mutagenesis was used to evaluate the full complement of natural amino acids at these positions, and resulted in the isolation of enzymes with catalytic rates comparable to those exhibited by the fastest mutants obtained via directed evolution. The substrate selectivities of enzymes resulting from random mutagenesis, DNA shuffling, and combinatorial saturation mutagenesis were evaluated using a series of distinct electrophiles. The results revealed that promiscuous substrate activities arose in a stochastic manner, as they did not correlate with catalytic efficiency towards the CMAC selection substrate. In contrast, chimeric enzymes previously constructed by homology-independent recombination of hGSTT-1 and rGSTT2-2 exhibited very different substrate promiscuity profiles, and showed a more defined relationship between evolved and promiscuous activities.

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“…The GST activities of the lysates were analyzed with eight alternative substrates. The measurements were made at 30°C in microplates on a SpectraMax Plus 384 microplate spectrophotometer (Molecular Devices), except with MCB, which was assayed fluorometrically in a microplate on a Fluoroskan Ascent (Labsystems) at 23°C (16). All measurements were made in duplicate within 12 h of thawing the lysates.…”

Section: Methodsmentioning

confidence: 99%

Functionally diverging molecular quasi-species evolve by crossing two enzymes

Emrén

Kurtovic²,

Runarsdottir³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Molecular evolution is frequently portrayed by structural relationships, but delineation of separate functional species is more elusive. We have generated enzyme variants by stochastic recombinations of DNA encoding two homologous detoxication enzymes, human glutathione transferases M1-1 and M2-2, and explored their catalytic versatilities. Sampled mutants were screened for activities with eight alternative substrates, and the activity fingerprints were subjected to principal component analysis. This phenotype characterization clearly identified at least three distributions of substrate selectivity, where one was orthogonal to those of the parent-like distributions. This approach to evolutionary data mining serves to identify emerging molecular quasi-species and indicates potential trajectories available for further protein evolution. directed evolution ͉ DNA shuffling ͉ glutathione transferase ͉ library ͉ multivariate analysis M olecular evolution is based on rearranged polynucleotide sequences and point mutations, which in turn give rise to novel phenotypes manifested as altered structures and functions. Biological mechanisms of genetic recombinations have counterparts in DNA shuffling and similar techniques for in vitro evolution (1). For evolution to occur, the mutants arising from progenitors undergo a Darwinian selection of the fittest. This functional filtering of the offspring in combination with structural boundary conditions governs the pathways of evolutionary change. Directed evolution similarly aims at gratifying qualities such as enhanced catalytic efficiency or increased thermal stability (2). Theoretical and experimental studies indicate that evolution operates on ensembles of mutants with a stochastic distribution of structural and functional properties rather than on single individuals that fit the prevailing conditions. These evolving units can be considered molecular ''quasi-species'' (3). Here, we show how such divergent subpopulations can be identified by screening and analysis of functional data by multivariate methods, illustrating the emergence of new distributions of functional properties in enzyme evolution.We approached molecular evolution by analyzing a library of enzyme variants obtained by recombination of DNA from two homologous glutathione transferases, GST M1-1 and GST M2-2 (4). The GSTs belong to a large family of enzymes that catalyze the conjugation of the nucleophilic tripeptide glutathione with a wide variety of genotoxic substrates. These conjugation reactions are prominent in the cellular inactivation of electrophilic compounds and promote the excretion of toxicants (5). Facile adaptation of substrate selectivities would appear to have selective advantage in particular for enzymes such as GSTs, which mount a protective response to a wide variety of toxic challenges. Results and DiscussionEnzyme Variants Represented in a Multidimensional Substrate-Activity Space. A library of mutant enzymes was produced by shuffling of cDNAs encoding human GST M1-1 (M1) and GST M2-2 (M2), ...

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Screening for recombinant glutathione transferases active with monochlorobimane

Cited by 23 publications

References 14 publications

The evaluation of novel natural products as inhibitors of human glutathione transferase P1-1

The evaluation of novel natural products as inhibitors of human glutathione transferase P1-1

The Evolution of Catalytic Efficiency and Substrate Promiscuity in Human Theta Class 1-1 Glutathione Transferase

Functionally diverging molecular quasi-species evolve by crossing two enzymes

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