Journal of General Microbiology (1991), 137, 2505-25 15. Printed in Great Britain 2505 The aconitase of Escherichia c o k purification of the enzyme and molecular cloning and map location of the gene (am) CHRISOSTOMOS PRODROMOU, MEGAN J. HAYNES and JOHN R. GUEST* The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Shefield, PO Box 594, Firth Court, Western Bank, Shefield SIO 2UH, UK (Received 24 April 1991; revised I0 July 1991; accepted 24 July 1991) The aconitase of Escherichia cofi was purified to homogeneity, albeit in low yield (0.6%). It was shown to be a monomeric protein of Mr 95000 or 97500 by gel filtration and SDS-PAGE analysis, respectively. The N-terminal amino acid sequence resembled that of the Baciflus subtifis enzyme (cifB product), but the similarity at the DNA level was insufficient to allow detection of the E. cofi acn gene using a 456 bp cifB probe. Phages containing the acn gene were isolated from a A-E. cofi gene bank by immunoscreening with an antiserum raised against purified bacterial enzyme. The acn gene was located at 28 min (1350 kb) in the physical map of the E cofi chromosomeby probing Southern blots with a fragment of the gene. Attempts to locate the gene using the same procedure with oligonucleotideprobes encoding segments of the N-terminal amino acid sequence were complicated by the lack of probe specificity and an inaccuracy in the physical map of Kohara ef d (Cef150, 495-508, 1987). Aconitase specific activity was amplified some 20-200-fold in cultures transformed with pGS447, a derivative of pUC119 containing the acn gene, and an apparent four-fold activation-deactivationof the phagemid-encoded enzyme was observed in late exponential phase. The aconitase antiserum cross-reacted with both the porcine and Salmonella typhimurium (Mr 120000) enzymes. Introduction Aconitase or citrate (isocitrate) hydro-lyase (EC 4.2.1 .3) functions in both the citric acid and glyoxylate cycles. It is a dehydratase-hydratase which catalyses the reversible isomerization of citrate and isocitrate via cis- aconitate. The mitochondrial aconitases have been purified from a variety of sources and shown to be monomeric proteins with an M, of about 79000-83000: porcine heart (Villafranca & Mildvan, 1971; Zheng et al., 1990); bovine heart (Ryden et al., 1984); and Saccharomyces cerevisiae (Scholze, 1983 ; Gangloff et al., 1990). In contrast, the fluoroacetate-induced enzyme from Candida lipolytica seems to be smaller, M, 68500 (Suzuki et al., 1975), and the Bacillus subtilis enzyme is considerably larger, M, 120000(Dingman & Sonenshein, 1987). Aconitase is interesting because it contains a cubane- like [4Fe-4S] cluster which is redox inactive, and essential for activity. Indeed, one of the Fe atoms (designated Fe,), has been shown to coordinate with the middle carboxyl group, and possibly the hydroxyl group, of the substrate (Kennedy et al., 1987; Werst et al., Abbreviations: ICDH, isocitrate dehydrogenase; TC, Tris/citrate. 0001-6896 0 1991 SGM 1990 a, 6). Exposure to oxygen during purification results in the loss of the Fe, atom, producing a catalytically inactive enzyme with a [3Fe-4S]+ cluster. However, the enzyme can be partially protected by adding citrate during purification, or reactivated by Fe2+ under reducing conditions to give a [4Fe-4SI2+cluster (Emp- tage et al., 1983; Kennedy et al., 1983; Kennedy & Bienert, 1988). The structures of the inactive [3Fe-4S] and active [4Fe-4S] forms of the porcine heart enzyme have been solved at 0.21 and 0.25 nm resolution, respectively (Robbins & Stout, 1989a, 6). The gene encoding the mitochondrial aconitase of S. cerevisiae has recently been cloned and the primary structure of the enzyme has been deduced from its nucleotide sequence (Gangloff et al., 1990). Similarly, the amino acid sequence of porcine heart aconitase has been deduced from the nucleotide sequence of the correspond- ing cDNA (Zheng et al., 1990)and the N-terminal region of the B. subtilis enzyme has been deduced from a segment of the citB gene (Rosenkrantz et al., 1985; Dingman & Sonenshein, 1987). Very little is known about the Escherichia coli aconitase and studies on the aconitase gene (am) have been hindered by the lack of mutants deficient in aconitase. Genes encoding all of the citric acid cycle