Human theta class glutathione transferase: the crystal structure reveals a sulfate-binding pocket within a buried active site

Rossjohn, Jamie; McKinstry, William J.; Oakley, Aaron J.; Verger, Denis; Flanagan, Jack U.; Chelvanayagam, Gareth; Tan, Kian L.; Board, Philip G.; Parker, Michael W.

doi:10.1016/s0969-2126(98)00034-3

Cited by 142 publications

(161 citation statements)

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“…Position 9 of the consensus sequence is a serine, but the corresponding position is Ala-122 in Ure2p. In the known structures of theta class GSTs (38,44,45), this serine's O␥ atom is either positioned, or may be positioned through a simple change of rotamer, to form a hydrogen bond with the sulfhydryl group of bound glutathione (GSH). This interaction has been proposed to stabilize the GSH as a thiolate anion, and thus activate GSH for the nucleophilic addition of the GSH sulfur atom to electrophilic groups of hydrophobic substrates (43).…”

Section: Resultsmentioning

confidence: 99%

“…This interaction has been proposed to stabilize the GSH as a thiolate anion, and thus activate GSH for the nucleophilic addition of the GSH sulfur atom to electrophilic groups of hydrophobic substrates (43). Mutation of this catalytic serine, Ser-11, to alanine in the A. thaliana theta class GST not only dramatically reduced activity to less than 0.5% the wild-type enzyme, but also significantly reduced the mutant's affinity for immobilized GSH (45). In the other GST classes, the hydroxyl group of a conserved tyrosine serves to stabilize the thiolate anion.…”

Section: Resultsmentioning

confidence: 99%

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The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p

Umland¹

2001

Proceedings of the National Academy of Sciences

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The yeast nonchromosomal gene [URE3] is due to a prion form of the nitrogen regulatory protein Ure2p. It is a negative regulator of nitrogen catabolism and acts by inhibiting the transcription factor Gln3p. Ure2p residues 1-80 are necessary for prion generation and propagation. The C-terminal fragment retains nitrogen regulatory activity, albeit somewhat less efficiently than the full-length protein, and it also lowers the frequency of prion generation. The crystal structure of this C-terminal fragment, Ure2p(97-354), at 2.3 Å resolution is described here. It adopts the same fold as the glutathione S-transferase superfamily, consistent with their sequence similarity. However, Ure2p(97-354) lacks a properly positioned catalytic residue that is required for S-transferase activity. Residues within this regulatory fragment that have been indicated by mutational studies to influence prion generation have been mapped onto the three-dimensional structure, and possible implications for prion activity are discussed.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p

Umland¹

2001

Proceedings of the National Academy of Sciences

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“…residue near its active site (46,47). That class of the enzyme has poor CDNB conjugating activity and does not adhere to GSH affinity matrices, but uniquely exhibits sulfatase activity with 1-menaphthylsulfate as a substrate.…”

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confidence: 99%

“…That class of the enzyme has poor CDNB conjugating activity and does not adhere to GSH affinity matrices, but uniquely exhibits sulfatase activity with 1-menaphthylsulfate as a substrate. For the class Theta enzyme it was proposed that electrostatic interactions between the Arg 107 residue and the intermediate leaving sulfate group could be important for activity (47). It should be noted that the rate-limiting step for CDNB conjugation catalyzed by a blowfly class Theta enzyme which lacks an active-site tyrosine residue was also shown to be viscosity-dependent (48).…”

mentioning

confidence: 99%

The Enhanced Affinity for Thiolate Anion and Activation of Enzyme-bound Glutathione Is Governed by an Arginine Residue of Human Mu Class Glutathione S-Transferases

Patskovsky

Patskovska

Listowsky

2000

Journal of Biological Chemistry

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A series of chimeric human Mu class glutathione Stransferases were designed to determine mechanisms by which they activate enzyme-bound glutathione (GSH) for reaction with electrophilic substrates. and Tyr 6 residues are required for thiolate anion formation and stabilization. The three-dimensional structure of ligand-free hGSTM2-2 determined by x-ray crystallography suggests that Arg 107 maintains an electrostatic interaction with the Asp 161 side chain (3 Å apart), but is distant from the GSH-binding site. However, an alternative energetically favorable model places the guanidino group 4 Å from the sulfur atom of bound GSH. It is suggested therefore, that in solution, motion of the positively charged arginine into the catalytic pocket could provide a counter ion to promote ionization of the sulfhydryl group of GSH, thereby accounting for the observed greater affinity of enzymes containing Arg 107 for binding of thiolate anion.

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“…3A), such that amino acids 1-231 are derived from the rat enzyme, whereas the final 13 amino acids correspond to residues 228-240 of the human GST. Modeling based on the homologous theta-class hGSTT2-2 crystal structure (27) indicated that the majority of the Cterminal helix of RH-A5 is derived from hGSTT1-1. Similarly, ITCHY clone RH-F2 contained a single 3Ј C͞O (Fig.…”

Section: Resultsmentioning

confidence: 99%

Evolution of highly active enzymes by homology-independent recombination

Griswold

Kawarasaki

Ghoneim

et al. 2005

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

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The theta-class GST enzymes hGSTT1-1 (human GST -1-1) and rGSTT2-2 (rat GST -2-2) share 54.3% amino acid identity and exhibit different substrate specificities. Homology-independent techniques [incremental truncation for the creation of hybrid enzymes (ITCHY) and SCRATCHY] and low-homology techniques (recombination-dependent exponential amplification PCR) were used to create libraries of chimeric enzymes containing crossovers (C͞Os) at positions not accessible by DNA family shuffling. Highthroughput flow cytometric screening using the fluorogenic rGSTT2-2-specific substrate 7-amino-4-chloromethyl coumarin led to the isolation of active variants with either one or two C͞Os. One of these enzymes, SCR23 (83% identity to hGSTT1-1), was encoded by a gene that exchanged helices 4 and 5 of hGSTT1-1 with the corresponding sequence from rGSTT2-2. Compared with either parent, this variant was found to have an improved k cat with the selection substrate and also exhibited activity for the conjugation of glutathione to ethacrynic acid, a compound that is not recognized by either parental enzyme. These results highlight the power of combinatorial homology-independent and lowhomology recombination methods for the generation of unique, highly active enzymes and also suggest a possible means of enzyme ''humanization.'' enzyme engineering ͉ enzyme humanization ͉ GST ͉ ITCHY ͉ high-throughput screening G STs play a crucial role in cellular detoxification by conjugating reactive electrophilic compounds to the tripeptide glutathione (GSH) (1). Mammals encode at least seven distinct classes of GSTs. The theta enzymes represent the oldest class of GSTs and, as such, are produced by a surprisingly diverse array of animals, plants, algae, and bacteria (2). Although theta-class enzymes retain the highly conserved GST 3D fold, they possess a characteristic C-terminal ␣-helical extension, which, unlike that of some alpha-class enzymes, covers both the electrophile and GSH-binding sites, thus clearly differentiating the theta class from other members of the soluble GST superfamily (3).The rat GST -2-2 (rGSTT2-2, 244 aa) and human GST -1-1 (hGSTT1-1, 240 aa) enzymes exhibit 54.3% overall amino acid identity. The GSH-binding domain, or G site, of the two enzymes (residues Ϸ1-77) is conserved (79.2% amino acid identity), and the 5Ј regions that encode this domain (nucleotides 1-231) exhibit 74.5% DNA sequence identity. In contrast, the electrophilic substrate binding domain, or H site (amino acid Ϸ89 to termini), of the rat and human enzymes (encoded by nucleotides Ϸ265-732 in rGSTT2-2 and nucleotides Ϸ265-720 in hG-STT1-1) exhibit only 41.4% amino acid identity and 57.9% DNA sequence identity. The sequence divergence of the two enzymes in the C-terminal domain is manifest in their respective electrophilic substrate selectivities. The rat enzyme preferentially catalyzes the GSH conjugation of 1-menaphythyl sulfate, whereas hGSTT1-1 exhibits a characteristic reactivity with dichloromethane (2). Additionally, we recently showed that rG-STT2-2 e...

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Human theta class glutathione transferase: the crystal structure reveals a sulfate-binding pocket within a buried active site

Cited by 142 publications

References 50 publications

The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p

The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p

The Enhanced Affinity for Thiolate Anion and Activation of Enzyme-bound Glutathione Is Governed by an Arginine Residue of Human Mu Class Glutathione S-Transferases

Evolution of highly active enzymes by homology-independent recombination

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