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The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase.

ABSTRACT: 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.

SUBMITTER: Griswold KE

PROVIDER: S-EPMC1995603 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase.

Griswold Karl E KE Aiyappan Nandini S NS Iverson Brent L BL Georgiou George G

Journal of molecular biology 20060909 3

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 sc ...[more]

PMID: 17011574

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Project description:Chloroplasts are organelles subjected to extreme oxidative stress conditions. Biomolecules produced in the chloroplasts act as signals guiding plant metabolism toward stress tolerance and play a major role in regulating gene expression in the nucleus. Herein, we used transplastomic plants as an alternative approach to expression of transgenes in the nucleus for conferring stress tolerance to abiotic stresses and herbicides. To investigate the morphophysiological and molecular mechanisms and the role of plastid expressed GSTs in tobacco stress detoxification and stress tolerance, we used transplastomic tobacco lines overexpressing a theta class glutathione transferase (GST) in chloroplasts. The transplastomic plants were tested under drought (0, 100, and 200 mM mannitol) and salinity (0, 150, and 300 mM NaCl) in vitro, and under herbicide stress (Diquat). Our results suggest that pt AtGSTT lines were tolerant to herbicide-induced oxidative and salinity stresses and showed enhanced response tolerance to mannitol-induced osmotic stress compared to WT plants. Overexpression of the Arabidopsis thaliana AtGSTT in the chloroplasts resulted in enhanced photo-tolerance and turgor maintenance under stress. Whole-genome transcriptome analysis revealed that genes related to stress tolerance, were upregulated in pt AtGSTT2a line under both control and high mannitol stress conditions. Transplastomic plants overexpressing the pt AtGSTT2a in the chloroplast showed a state of acclimation to stress, as only limited number of genes were upregulated in the pt AtGSTT2a transplastomic line compared to WT under stress conditions while at the same time genes related to stress tolerance were upregulated in pt AtGSTT2a plants compared to WT in stress-free conditions. In parallel, the metabolic profile indicated limited perturbations of the metabolic homeostasis in the transplastomic lines and greater accumulation of mannitol, and soluble sugars under high mannitol stress. Therefore, transplastomic lines seem to be in a state of acclimation to stress under stress-free conditions, which was maintained even under high mannitol stress. The results help to elucidate the role of GSTs in plant abiotic stress tolerance and the underlying mechanisms of the GSTs expressed in the chloroplast, toward environmental resilience of cultivated crops.

Dataset Information

The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase.

Publications

The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase.

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