Biochem

Biochem. J. (2006) 398 (451–460) (Printed in Great Britain)

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
Jaekwang KIM1, Hyunsuk SUH1, Songhee KIM, Kiyoung KIM, Chiyoung AHN2 and Jeongbin YIM3
School of Biological Sciences, Seoul National University, Seoul151-742, Korea
________________________________________
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 drosopterinbiosynthesis. 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 relativelyhigh 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 ofthe 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. Inconclusion, 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.
________________________________________
Key words: Drosophila, drosopterin, eye colour mutant, glutathione S-transferase, pyrimidodiazepine synthase, sepia.
Abbreviations used: CDNB, 1-chloro-2,4-dinitrobenzene; CG, computed gene; DHA,dehydroascorbate; HED, b-hydroxyethylene disulfide; GMR-gal4, ‘glass multiple reporter’ promoter-dependent gal4 construct/transgene; GST, glutathione S-transferase; GSTO1, Omega class GST 1; ORF, open reading frame; PDA, 6-acetyl-2-amino-3,7,8,9-tetrahydro-4H-pyrimido[4,5-b]-[1,4]diazepin-4-one or pyrimidodiazepine; 6-PTP, 2-amino-4-oxo-6-pyruvoyl-5,6,7,8-tetrahydropteridine; H2NTP,2-amino-4-oxo-6-(D-erythro-1´,2´,3´-trihydroxypropyl)-7,8-dihydropteridine triphosphate; poly(A)+ RNA, polyadenylated RNA; RT, reverse transcriptase; UAS, upstream activation sequence.
1These authors have contributed equally to this work.
2Present address: Cell and Tissue Engineering Products Team, Biologics Headquarters, Korea Food and Drug Administration, Seoul 122-704, Korea.
3To whom correspondence shouldbe addressed (email jyim@snu.ac.kr).
________________________________________
INTRODUCTION
The eye colour pigments of Drosophila melanogaster have been the subject of many investigations since the discovery of the first eye colour mutant. Two classes of pigments, the brown ‘ommochromes’ and red ‘drosopterins’, are responsible for the typical eye colour of Drosophila. Drosopterins consist of at...