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

Abstract

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.

Figure 1. adgst1AS1 and gstD5 gene organization of 8A.2 clone

The gstD5 gene is located 4620 bp upstream, with the opposite orientation to adgst1AS1. The 15694 bp genomic sequence has GenBank® accession number AF251478.

Figure 2. The gstD5 gene organization is composed of three coding regions that code for one GST protein

The coding sequence is shown in bold, with the translated amino acid sequence below. The last codon of exon 1 is split, with the last base of the codon in exon 2.

Figure 3. Amino acid sequence comparison of A. dirus adGSTD5-5 and the orthologous enzyme of AgGSTD7 from A. gambiae (agambD7) and five previously obtained A. dirus Delta GSTs using GeneDoc version 2.6

The sequences are adGSTD1-1, adGSTD2-2, adGSTD3-3, adGSTD4-4 and adGSTD6-6 (GenBank® accession numbers AF273041, AF273038, AF273039, AF273040 and AY014406 respectively [6,8]). (A) The dashes indicate gaps in the sequence. Black shading indicates 100% conserved residues for the seven sequences, dark grey indicates 80% conserved, and light grey indicates 60% conserved. (B) Matrix table of percentage amino acid identities (top value) and percentage amino acid similarities (bottom value) for the sequences aligned in (A).

Figure 4. Sequence of two putative promoters of gstD5 (Ad5-923 and Ad5-3429)

Potential transcription-factor-binding sites were mapped on the region of these two putative promoters by the MatInspector program. BCD, bicoid; ELF, grainyhead protein of Drosophila; ER, oestrogen receptor; HFH8, forkhead domain protein; SRY, sex-determining region Y; VBP, vitellogenin-binding protein. The binding site of each potential transcription factor is underlined or overlined. The (+) indicates the binding site on the sense strand, while (−) indicates the binding site on the antisense strand.

Figure 5. Fatty acid effects on adGSTD5-5 CDNB activity

The final concentration of fatty acids tested was 50 μM in the standard CDNB assay. Data show the changed enzyme activity for CDNB in the presence of the respective fatty acid, where negative values show inhibition and positive values show activation relative to the absence of any fatty acid.

Figure 6. Stereo view of adGSTD5-5

The view is looking down on the 2-fold axis into the active site, with the glutathione sulphonic acid shown as a ball-and-stick figure. The co-ordinates for the tertiary structure have been deposited in the Protein Data Bank under the accession code 1R5A.

Figure 7. Cartoon representation of adGSTD5-5

Cylinders represent helices, arrows represent strands, and lines represent random coil. Left: adGSTD5-5, with secondary structure elements labelled. Right: Aligned Anopheles GSTs. adGSTD3-3 is in blue, adGSTD4 is in red, and adGSTD5-5 is in yellow.

Figure 8. Stereo views of the electrostatic potential surface of the active-site pockets of adGSTD5-5 and adGSTD3-3

The two tertiary structures were aligned to illustrate the same view of the active site. The top panel shows adGSTD5-5, and the bottom panel shows adGSTD3-3. A yellow ball-and-stick representation shows the GSH in the pocket.