Identification, characterization and structure of a new Delta class glutathione transferase isoenzyme

The insect GST (glutathione transferase) supergene family encodes a varied group of proteins belonging to at least six individual classes. Interest in insect GSTs has focused on their role in conferring insecticide resistance. Previously from the mosquito malaria vector Anopheles dirus, two genes encoding five Delta class GSTs have been characterized for structural as well as enzyme activities. We have obtained a new Delta class GST gene and isoenzyme from A. dirus, which we name adGSTD5-5. The adGSTD5-5 isoenzyme was identified and was only detectably expressed in A. dirus adult females. A putative promoter analysis suggests that this GST has an involvement in oogenesis. The enzyme displayed little activity for classical GST substrates, although it possessed the greatest activity for DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] observed for Delta GSTs. However, GST activity was inhibited or enhanced in the presence of various fatty acids, suggesting that the enzyme may be modulated by fatty acids. We obtained a crystal structure for adGSTD5-5 and compared it with other Delta GSTs, which showed that adGSTD5-5 possesses an elongated and more polar active-site topology.

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

Structure ◽

10.1016/s0969-2126(98)00034-3 ◽

1998 ◽

Vol 6 (3) ◽

pp. 309-322 ◽

Cited By ~ 105

Author(s):

Jamie Rossjohn ◽

William J McKinstry ◽

Aaron J Oakley ◽

Denis Verger ◽

Jack Flanagan ◽

...

Keyword(s):

Crystal Structure ◽

Active Site ◽

Glutathione Transferase ◽

Binding Pocket

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Clarification of the role of key active site residues of glutathione transferase Zeta/maleylacetoacetate isomerase by a new spectrophotometric technique

Biochemical Journal ◽

10.1042/bj20030625 ◽

2003 ◽

Vol 374 (3) ◽

pp. 731-737 ◽

Cited By ~ 32

Author(s):

Philip G. BOARD ◽

Matthew C. TAYLOR ◽

Marjorie COGGAN ◽

Michael W. PARKER ◽

Hoffman B. LANTUM ◽

...

Keyword(s):

Crystal Structure ◽

Active Site ◽

Glutathione Transferase ◽

Salt Bridge ◽

Side Chain ◽

Active Site Residues ◽

Conserved Residues ◽

Isomerase Activity ◽

Catalytic Role

hGSTZ1-1 (human glutathione transferase Zeta 1-1) catalyses a range of glutathione-dependent reactions and plays an important role in the metabolism of tyrosine via its maleylacetoacetate isomerase activity. The crystal structure and sequence alignment of hGSTZ1 with other GSTs (glutathione transferases) focused attention on three highly conserved residues (Ser-14, Ser-15, Cys-16) as candidates for an important role in catalysis. Progress in the investigation of these residues has been limited by the absence of a convenient assay for kinetic analysis. In this study we have developed a new spectrophotometric assay with a novel substrate [(±)-2-bromo-3-(4-nitrophenyl)propionic acid]. The assay has been used to rapidly assess the potential catalytic role of several residues in the active site. Despite its less favourable orientation in the crystal structure, Ser-14 was the only residue found to be essential for catalysis. It is proposed that a conformational change may favourably reposition the hydroxyl of Ser-14 during the catalytic cycle. The Cys16→Ala (Cys-16 mutated to Ala) mutation caused a dramatic increase in the Km for glutathione, indicating that Cys-16 plays an important role in the binding and orientation of glutathione in the active site. Previous structural studies implicated Arg-175 in the orientation of α-halo acid substrates in the active site of hGSTZ1-1. Mutation of Arg-175 to Lys or Ala resulted in a significant lowering of the kcat in the Ala-175 variant. This result is consistent with the proposal that the charged side chain of Arg-175 forms a salt bridge with the carboxylate of the α-halo acid substrates.

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