Biochem. J. (1986) 237, 427-437 (Printed in Great Britain) The cloning and expression of the aroL gene from Escherichia coli K12 Purification and complete amino acid sequence of shikimate kinase II, the aroL-gene product Gary MILLAR,* Ann LEWENDON,* Michael G. HUNTERt and John R. COGGINS*t *Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K., and tSearle Research and Development, P.O. Box 53, Lane End Road, High Wycombe, Bucks, HP12 4HL, U.K. The aroL gene encoding the enzyme shikimate kinase II was cloned from Escherichia coli K12. Construction of over-expressing strains permitted for the first time the purification to homogeneity of a monofunctional shikimate kinase. The complete amino acid sequence of shikimate kinase II was determined by a combined nucleotide and direct amino acid sequencing strategy. E. coli shikimate kinase II is a monomeric enzyme containing 173 amino acid residues with a calculated Mr 18 937. The amino acid sequence contains a region homologous with other kinases and ATP-requiring enzymes. Evidence is presented suggesting that the transcriptional start site of the aroL gene is located within a potential operator site. INTRODUCTION Shikimatekinase(EC2.7.1.71)catalysesthephosphoryl- ation of shikimic acid to yield shikimate 3-phosphate. This reaction is the fifth step of the early common pathway for aromatic amino acid biosynthesis (the shikimate pathway) in micro-organisms. One of the remarkable features of this pathway is the very different structural organization of the enzymes in bacteria and fungi. It has been shown that in Neurospora crassa the central five enzymes of the seven-step pathway occur as a multifunctional enzyme consisting of two identical pentafunctional polypeptide chains of Mr 165000 (Lumsden & Coggins, 1977, 1978; Gaertner & Cole, 1977; Smith & Coggins, 1983; Lambert et al., 1985). A similar multifunctional enzyme occurs in Saccharomyces cerevisiae (Larimer et al., 1983; K. Duncan, R. M. Edwards & J. R. Coggins, unpublished work) and in a number of other species of fungi (Strauss, 1979; Bode & Birnbaum, 1981; Kinghorn & Hawkins, 1982; Nakanishi & Yamamoto, 1984). In contrast, in Escherichia coli the corresponding enzymes are separable (Berlyn & Giles, 1969) and presumably monofunctional. Four of these monofunctional E. coli enzymes, 3-dehydroquinate synthase (Frost et al., 1984), 3-dehydroquinase (Chaud- huri et al., 1986), shikimate dehydrogenase (Chaudhuri & Coggins, 1985) and 5-enolpyruvylshikimate-3-phos- phate synthase (Lewendon & Coggins, 1983; Duncan et al., 1984), have been purified and characterized. As part of a detailed comparative study of the N. crassa and S. cerevisiae multifunctional enzymes with the five corres- ponding monofunctional E. coli enzymes we required to purify and characterize E. coli shikimate kinase. Two shikimate kinase activities have been detected in E. coli (Ely & Pittard, 1979) and in Salmonella typhimurium (Morell & Sprinson, 1968; Berlyn & Giles, 1969). Neither of the E. coli isoenzymes has been purified and in only one case, shikimate kinase II, has the gene (aroL) been located (Ely & Pittard, 1979). In Bacillus subtilis there is a single shikimate kinase, which is a component of a trifunctional multienzyme complex. This complex contains a bifunctional polypeptide carrying 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and chorismate mutase activities as well as a monofunc- tional shikimate kinase polypeptide (Nakatsukasa & Nester, 1972). The kinase polypeptide, which is active only in the complex, has been purified to homogeneity and has an Mr of 10000 (Huang et al., 1975). In B. subtilis the regulatory properties of shikimate kinase suggest that it may represent a crucial allosteric step in the shikimate pathway (Huang et al., 1975; Nasser et al., 1969). The reason for the existence of two shikimate kinase isoenzymes in E. coli is not known, but it has been suggested that shikimate may be a branch-point intermediate for two distinct pathways (Weiss & Edwards, 1980). The chromosomal location of the aroL gene is known (Ely & Pittard, 1979); it maps close to the proC and phoA genes at 9 min on the E. coli chromosome (Bachmann, 1983). The present paper reports the cloning and sequence of the E. coli aroL gene and the over-production, purification to homogeneity and N-terminal sequencing of the gene product, shikimate kinase II. MATERIALS AND METHODS Media and bacterial strains Cells were prepared by growth at 37 °C on minimal media (Maniatis et al., 1982) supplemented with 0.2% glucose containing, where appropriate, an antibiotic or nutritional supplement. Amino acid supplements were added at 50,ug/ml (final concentration). Bacterial strains used are listed in Table 1, and the plasmids used are listed in Table 2. Transformation of E. coli Competent cells were prepared and transformations carried out as described by Daghert & Ehrlich (1979). Abbreviations used: bp, base-pairs; f.p.l.c., fast protein liquid chromatography. t To whom correspondence should be addressed. Vol. 237 427