Albert J. Ketterman

The crystal structures of glutathione S-transferases isozymes 1-3 and 1-4 from Anopheles dirus species B.

Glutathione S‐transferases (GSTs) are dimeric proteins that play an important role in cellular detoxification. Four GSTs from the mosquito Anopheles dirus species B (Ad), an important malaria vector in South East Asia, are produced by alternate splicing of a single transcription product and were previously shown to have detoxifying activity towards pesticides such as DDT. We have determined the crystal structures for two of these alternatively spliced proteins, AdGST1–3 (complexed with glutathione) and AdGST1–4 (apo form), at 1.75 and 2.45 Å resolution, respectively. These GST isozymes show differences from the related GST from the Australian sheep blowfly Lucilia cuprina; in particular, the presence of a C‐terminal helix forming part of the active site. This helix causes the active site of the Anopheles GSTs to be enclosed. The glutathione‐binding helix α2 and flanking residues are disordered in the AdGST1–4 (apo) structure, yet ordered in the AdGST1–3 (GSH‐bound) structure, suggesting that insect GSTs operate with an induced fit mechanism similar to that found in the plant phi‐ and human pi‐class GSTs. Despite the high overall sequence identities, the active site residues of AdGST1–4 and AdGST1–3 have different conformations.

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.

DDT-resistance in Anopheles gambiae (Diptera: Culicidae) from Zanzibar, Tanzania, based on increased DDT-dehydrochlorinase activity of glutathione S-transferases

DDT-resistant Anopheles gambiae Giles from Zanzibar, Tanzania, had increased levels of DDT-dehydrochlorination compared to a DDT-susceptible strain. Glutathione S-transferases (GSTs) are responsible for conversion of DDT to DDE in both the susceptible and resistant strains. Sequential column chromatography, including Q-Sepharose, S-hexylglutathione agarose, hydroxylapatite and phenyl Sepharose, allowed the partial purification of seven GSTs. All seven GSTs possessed different degrees of DDTase activity. There was an eight-fold increase in total DDTase activity in the resistant compared to the susceptible enzymes. Characterization with three substrates, 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and DDT, revealed the different substrate specificity for each isolated GST indicating different isoenzymes. GST Va possessed 60% of total DDTase activity suggesting that it contributed most to DDT-metabolism in this insect species. The DDTase activity of the GSTs in both strains of A. gambiae were found to be correlated with the GST activities toward DCNB. Preliminary studies on DDT-resistant and susceptible A. gambiae showed that both DDT-resistance and the increased levels of GST activity were stage specific which suggested that different GSTs may be involved in DDT-resistance in adults and larvae of A. gambiae .

Purification and characterization of a major glutathione S-transferase from the mosquito Anopheles dirus (Species B)

The major form of glutathione S-transferase (GST) activity from the mosquito Anopheles dirus (species B), a vector of malaria in Thailand has been purified 421-fold. It constituted approx. 20% of the total measured CDNB conjugating activity in the homogenate. This enzyme appeared as a single band of 25.0 +/- 0.26 kDa on SDS-PAGE and was kinetically characterized with 10 substrates and 4 inhibitors. The enzyme is capable of catalysing dehydrochlorination of 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) in vitro at a rate of 4.4 nmol of 1,1-dichloro-2,2-bis-(p-chlorophenyl)ethane (DDE) formation per mg protein. This is comparable to the rate of catalysis of the orthologous isoenzyme from An. gambiae reported previously. The IC50 plots of the inhibitor data (fractional velocity vs log [I]) for three of the inhibitors indicate the homogenous nature of this enzyme. However, inhibition by ethacrynic acid demonstrates more than a single affinity site for interaction. The six N-terminal amino acids of the purified enzyme are identical to a GST reported from Aedes aegypti, which was indicated to play a role in DDT-resistance in this species. The results suggest that the two enzymes may belong to the same class, however each possesses a different specificity.

Insect glutathione transferases.

This article is an overview of the current knowledge of insect glutathione transferases. Three major topics are discussed: the glutathione transferase contributions to insecticide resistance, the polymorphic nature of the insect glutathione transferase superfamily, and a summary of the current structure-function studies on insect glutathione transferases.

Heterologous expression and characterization of alternatively spliced glutathione S-transferases from a single Anopheles gene.

Three cDNA sequences of glutathione S-transferase (GST), adgst1-2, adgst1-3 and adgst1-4, which are alternatively spliced products of the adgst1AS1 gene, were obtained from fourth instar larvae of Anopheles dirus mosquito by reverse transcriptase PCR reactions. The nucleotide sequences of these three cDNAs share >67% identity and the translated amino acid sequences share 61-64% identity. A comparison of the An. dirus to the An. gambiae enzymes shows that adGST1-2 versus agGST1-4, adGST1-3 versus agGST1-5 and adGST1-4 versus agGST1-3 have 85, 92 and 85% amino acid sequence identity, respectively, which confirms that orthologous isoenzymes occur across anopheline species. These three proteins were expressed at high levels, approximately 15-20 mg from 200 ml of E. coli culture. The recombinant enzymes were purified by affinity chromatography on an S-hexylglutathione agarose column. The subunit sizes of adGST1-2, adGST1-3 and adGST1-4 are 24.3, 23.9 and 25.1 kDa. The recombinant enzymes have high activities with 1-chloro-2,4-dinitrobenzene (CDNB), detectable activity with 1,2-dichloro-4-nitrobenzene but markedly low activity with ethacrynic acid and p-nitrophenethyl bromide. adGST1-3 was shown to be the most active enzyme from the kinetic studies. Permethrin inhibition of CDNB activity, at varying concentrations of CDNB, was significantly different, being uncompetitive for adGST1-2, noncompetitive for adGST1-3 and competitive for adGST1-4. In contrast, permethrin inhibition with varying glutathione concentrations was noncompetitive for all three GSTs. Despite the enzymes being splicing products of the same gene and sharing identical sequence in the N-terminal 45 amino acids, these GSTs show distinct substrate specificities, kinetic properties and inhibition properties modulated by the differences in the C-terminus.

Isoenzymes of glutathione S-transferase from the mosquito Anopheles dirus species B: the purification, partial characterization and interaction with various insecticides

Previously we have purified and characterized a major glutathione S-transferase (GST) activity, GST-4a, from the Thai mosquito Anopheles dirus B, a model mosquito for study of anopheline malaria vectors [Prapanthadara, L. Koottathep, S., Promtet, N., Hemingway, J. and Ketterman, A.J. (1996) Insect Biochem. Mol. Biol. 26:3, 277-285]. In this report we have purified an isoenzyme, GST-4c, which has the greatest DDT-dehydrochlorinase activity. Three additional isoenzymes, GST-4b, GST-5 and GST-6, were also partially purified and characterized for comparison. All of the Anopheles GST isoenzymes preferred 1-chloro-2,4-dinitrobenzene (CDNB) as an electrophilic substrate. In kinetic studies with CDNB as an electrophilic substrate, the V(max) of GST-4c was 24.38 micromole/min/mg which was seven-fold less than GST-4a. The two isoenzymes also possessed different K(m)s for CDNB and glutathione. Despite being only partially pure GST-4b had nearly a four-fold greater V(max) for CDNB than GST-4c. In contrast, GST-4c possessed the greatest DDT-dehydrochlorinase specific activity among the purified insect GST isoenzymes and no activity was detected for GST-5. Seven putative GST substrates used in this study were not utilized by An. dirus GSTs, although they were capable of inhibiting CDNB conjugating activity to different extents for the different isoenzymes. Bromosulfophthalein and ethacrynic acid were the most potent inhibitors. The inhibition studies demonstrate different degrees of interaction of the An. dirus isoenzymes with various insecticides. The GSTs were inhibited more readily by organochlorines and pyrethroids than by the phosphorothioates and carbamate. In a comparison between An. dirus and previous data from An. gambiae the two anopheline species possess a similar pattern of GST isoenzymes although the individual enzymes differ significantly at the functional level. The available data suggests there may be a minimum of three GST classes in anopheline insects.

A preliminary characterization of the cytosolic glutathione transferase proteome from Drosophila melanogaster

The cytosolic GST (glutathione transferase) superfamily has been annotated in the Drosophila melanogaster genome database. Of 36 genes, four undergo alternative splicing to yield a total of 41 GST proteins. In the present study, we have obtained the 41 transcripts encoding proteins by RT (reverse transcription)-PCR using RNA template from Drosophila S2 cells, an embryonic cell line. This observation suggests that all of the annotated DmGSTs (D. melanogaster GSTs) in the proteome are expressed in the late embryonic stages of D. melanogaster. To avoid confusion in naming these numerous DmGSTs, we have designated them following the universal GST nomenclature as well as previous designations that fit within this classification. Furthermore, in the cell line, we identified an apparent processed pseudogene, gste8, in addition to two isoforms from the Delta class that have been published previously. Only approximately one-third of the expressed DmGSTs could be purified by conventional GSH affinity chromatography. The diverse kinetic properties as well as physiological substrate specificity of the DmGSTs are such that each individual enzyme displayed a unique character even compared with members from the same class.

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.

Purification and characterization of two human liver carboxylesterases

1. Two carboxylesterases (EC 3.1.1.1) purified from human livers were distinguished by pI (isoelectric point), nondenaturing polyacrylamide gel electrophoresis, molecular weight, catalytic activity, N-terminus and immunological cross-reactivity. 2. The low pI carboxylesterase has not been reported previously. 3. Numerous bands seen when each enzyme was focused on analytical IEF gels could not be separated. 4. When sections of the band pattern was refocused, the original complete band pattern was generated. 5. Both the mid and low pI carboxylesterases had catalytic activity for xenobiotics as well as medium and long chain fatty acid esters.