Single amino acid changes outside the active site significantly affect activity of glutathione S-transferases.

Glutathione S-transferases (GSTs: E.C. 2.5.1.18) are a multigene family of multifunctional dimeric proteins that play a central role in detoxication. Four allelic forms of the mosquito Anopheles dirus GST, adGST1-1, were cloned, expressed and characterized. The one or two amino acid changes in each allelic form was shown to confer different kinetic properties. Based on an available crystal structure, several of the residue changes were not in the putative substrate-binding pocket. Modeling showed that these insect Delta class GSTs also possess a hydrophobic surface pocket reported for Alpha, Mu and Pi class GSTs. The atom movement after replacement and minimization showed an average atom movement of about 0.1 A for the 0 to 25 A distance from the alpha carbon of the single replaced residue. This does not appear to be a significant movement in a static modeled protein structure. However, 200-500 atoms were involved with movements greater than 0.2 A. Dynamics simulations were performed to study the effects this phenomenon would exert on the accessible conformations. The data show that residues affecting nearby responsive regions of tertiary structure can modulate enzyme specificities, possibly through regulating attainable configurations of the protein.

Genomic organization and putative promoters of highly conserved glutathione S-transferases originating by alternative splicing in Anopheles dirus.

The genomic DNA of a GST class I alternative splicing gene has been characterized from Anopheles dirus, a Thai malaria vector. This gene organization is highly conserved in An. dirus and Anopheles gambiae (aggst1alpha), with >80% nucleotide identity in the coding region. Their gene organization contains six exons for four mature GST transcripts, which share exon 1 and exon 2 but vary between four different exon 3 sequences (exon 3A-3D). The deduced amino acid sequence of the GST transcripts from these two genes also shows very high conservation, with 85-93% identity for each orthologous gene. Two putative promoters and possible regulatory elements were predicted by a combination of the TSSW and MatInspector programs. The Ad214 promoter is proposed to be involved in developmental stage regulation. The Ad2112 promoter would appear to respond to intra- or extracellular stimuli. These two Anopheline species appear to have diverged in the distant past based on gene neighbors and phylogenetic data, yet these GST genes are still conserved. Therefore it is highly probable that this GST gene organization has one or more important roles.