Identification and characteristics of the structural gene for the Drosophila eye colour mutant sepia, encoding PDA synthase, a member of the Omega class glutathione S-transferases

Abstract

The eye colour mutant sepia (se1) is defective in PDA {6-acetyl-2-amino-3,7,8,9-tetrahydro-4H-pyrimido[4,5-b]-[1,4]diazepin-4-one or pyrimidodiazepine} synthase involved in the conversion of 6-PTP (2-amino-4-oxo-6-pyruvoyl-5,6,7,8-tetrahydropteridine; also known as 6-pyruvoyltetrahydropterin) into PDA, a key intermediate in drosopterin biosynthesis. However, the identity of the gene encoding this enzyme, as well as its molecular properties, have not yet been established. Here, we identify and characterize the gene encoding PDA synthase and show that it is the structural gene for sepia. Based on previously reported information [Wiederrecht, Paton and Brown (1984) J. Biol. Chem. 259, 2195-2200; Wiederrecht and Brown (1984) J. Biol. Chem. 259, 14121-14127; Andres (1945) Drosoph. Inf. Serv. 19, 45; Ingham, Pinchin, Howard and Ish-Horowicz (1985) Genetics 111, 463-486; Howard, Ingham and Rushlow (1988) Genes Dev. 2, 1037-1046], we isolated five candidate genes predicted to encode GSTs (glutathione S-transferases) from the presumed sepia locus (region 66D5 on chromosome 3L). All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro. The molecular mass of recombinant CG6781 was estimated to be 28 kDa by SDS/PAGE and 56 kDa by gel filtration, indicating that it is a homodimer under native conditions. Sequencing of the genomic region spanning CG6781 revealed that the se1 allele has a frameshift mutation from 'AAGAA' to 'GTG' at nt 190-194, and that this generates a premature stop codon. Expression of the CG6781 open reading frame in an se1 background rescued the eye colour defect as well as PDA synthase activity and drosopterins content. The extent of rescue was dependent on the dosage of transgenic CG6781. In conclusion, we have discovered a new catalytic activity for an Omega class GST and that CG6781 is the structural gene for sepia which encodes PDA synthase.

A Study of the Glutathione Transferase Proteome of Drosophila melanogaster: Use of S- Substituted Glutathiones as Affinity Ligands

“…The Omega isoform CG6673B has a high dehydroascorbate reductase activity (Kim et al, 2006) and accounts for the high reductase activity in the material eluted from these columns. These isoforms also have minimal catalytic activity with CDNB (Kim et al, 2006) suggesting that GSTs may bind to GSH conjugates of a substrate with which they have little or no catalytic activity.…”

“…The Omega isoform CG6673B has a high dehydroascorbate reductase activity (Kim et al, 2006) and accounts for the high reductase activity in the material eluted from these columns. These isoforms also have minimal catalytic activity with CDNB (Kim et al, 2006) suggesting that GSTs may bind to GSH conjugates of a substrate with which they have little or no catalytic activity. This is supported by the observation that the matrices carrying conjugates of CDNB and DCNB, substrates that discriminate strongly between differing GSTs, appear to bind almost identical groups of proteins, both GSTs and non-GSTs.…”

“…As in the previous experiments Sigma GST had not been completely removed and there was detectable presence of GSTs D1 and D3. In addition to the Omega GSTs revealed by the other two matrices, the use of NB-SG showed the presence of CG6781, the Omega GST involved in synthesis of eye pigments (Kim et al 2006). Table 4.…”

“…Assays with p-nitrophenol acetate (p-NPA) (Keen et al, 1976), trans-2-nonenal (TNE) (Brophy et al, 1989) and dehydroascorbic acid (DHA) (Kim et al, 2006) were also carried out to study the specificity of the partially purified GST preparations.…”

“…These include olfaction (Rogers et al, 1999), regulation of apoptosis (Adler et al, 1999;Udomsinprasert et al, 2004), eye pigment synthesis (Kim et al, 2006), haeme binding (Lumjuan et al, 2007) and wound healing (Li et al, 2002). In order to disentangle these multiple roles, it is desirable to develop methods to characterize as fully as possible the expression under differing conditions of the many insect glutathione transferases.…”