Molecular Microbiology (1991) 5(9). 2143-2152 Molecular cloning, characterization and analysis of the regulation of the AR02gene, encoding chorismate synthase, of Saccharomyces cerevisiae D. G. L. Jones, U. Reusser and G. H. Braus * Institute of Microbiology, Swiss Federai Institute of Technology (ETH), Schmeizbergstrasse 7, CH-8092 Zurich, Switzerland. Summary We describe here the cloning, characterization and analysis of the regulation of the ARO2 gene of Sac- charomyces cerevisiae, the first reported study of a eukaryotic gene encoding chorismate synthase (E.C. 4.6.1.4). The gene contains an ORF of 1128 bp, encod- ing a protein with a calculated molecular mass of 40.8 kDa. ARO2 is regulated under the general controi system' for amino acid biosynthesis by the transcrip- tional activator GCN4 which binds in vitro at three sites within the AR02 promoter. The AR02 gene product is highly similar to its Escherichia coli counterpart, with a 47% identity dis- tributed over the entire length of the peptide. We therefore suggest that the S. cerevisiae chorismate synthase, in contrast to the Neurospora crassa enzyme, but iike other chorismate synthases, is a monofunctional peptide, solely possessing choris- mate synthase activity. introduction The biosynthesis of aromatic amino acids in plants and microorganisms proceeds via the shikimic acid pathway, a seven-step enzymatic pathway resulting in the forma- tion of chorismic acid (Hasiam, 1974). Chorismic acid is a centrai precursor and key branch-point intermediate in aromatic amino acid biosynthesis as its distribution can be channelled towards either the production of tyrosine and phenylalanine or towards tryptophan. Furthermore, chorismic acid can also be channelled towards the pro- duction of other aromatic compounds, namely vitamin K, ^aminobenzoate, 2,3 dihydroxy-benzoate and ubiqui- none. The production of chorismic acid from its immediate precursor, 5-enolpyruvylshikimate-3-phosphate (EPSP), is catalysed by the enzyme chorismate synthase (5- ReceJved 16 April, 1991; revised 19 June. 1991. *For correspondence. Tel, (01) 256 3327; Fax (01) 252 9613. enolpyruvylshikimate-3-phosphate phospholyase, E.C. 4.6.1.4). The reaction, which involves a 1,4-rrans-elimina- tion of phosphoric acid from EPSP, is poorly understood owing to a stereochemical ambiguity (Hawkes et ai.. 1990) and no net change in the redox state occurring, despite an absoiute requirement for a reduced flavin cofactor (White etai. 1988). In order to better understand the mechanism and con- trol of this important reaction chcrismate synthase has been characterized from three bacterial sources, namely Escherichia coli {White etai. 1988; Charles etai, 1990), Bacillus subtilis (Hasan and Nester. 1978) and Salmonella typhi (Charles ef ai, 1990), a fungal source, Neurospora crassa (Welch et ai., 1974; White ef ai, 1988) and two plant sources, namely Pisum sativum (Mousdale and Coggins, 1986) and Corydaiis semper- virens (Schaller etai. t990). In the cases of E. co//and S. typhi the corresponding genes encoding the enzyme have been cloned and sequenced (White et ai. 1988; Charles ef ai.. 1990) and In the case of N. crassa the enzyme has been extensively purified (Welch ef ai, 1974; White efa/., 1988). It appears that in N. crassa the choris- mate synthase activity is intrinsically associated with a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent flavin reductase (diaphorase) activ- ity, i.e. it is a btfunctionai enzyme, whereas in E. coli, P. sativum. B. subtilis and C. sempervirens the enzyme is monofuncticnal, possessing only chorismate synthase activity and relying on a separate, external diaphorase activity. In the case of B. subtilis it appears that the diaphorase is a specific, tightly associated but neverthe- less separate polypeptide. It is possible that the other monofunctional chorismate synthases may rely on gen- eral diaphcrase activity throughout the cell to generate sufficient reduced flavin for the reaction. The shikimate and subsequent aromatic amino acid biosynthetic pathways of the yeast Saccharomyces cere- visiae are well understood and many of the genes encod- ing key enzymes in the process have been cloned, sequenced and the gene products characterized (reviewed In Jones and Fink, 1982; Braus, 1991). Further- more, the regulation of the relevant genes has been thoroughly investigated and for the majority has been shown to be strongly dependent on the well-studied