The aconitase of Escherichia coli 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 Bacillus subtilis enzyme (citB product), but the similarity at the DNA level was insufficient to allow detection of the E. coli acn gene using a 456 bp citB probe. Phages containing the acn gene were isolated from a λ-E. coli 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. coli chromosome by probing Southern blots with a fragment of the gene. Attempts to locate the gene using the same procedure with oligonucleotide probes 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 et al. (Cell 50, 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—deactivation of 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.
AndrewsS. C.,
HarrisonP. M.,
GuestJ. R.1989; Cloning, sequencing, and mapping of the bacterioferritin gene (bfr) of Escherichia coli K-12. Journal of Bacteriology 171:3940–3947
BradfordM. M.1976; A rapid and sensitive method for the quantitative determination of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
DingmanD. W.,
SonensheinA. L.1987; Purification of aconitase from Bacillus subtilis and correlation of its N-terminal amino acid sequence with the citB gene. Journal of Bacteriology 169:3062–3067
EmptageM. H.,
DreyerJ.-L.,
KennedyM. C.,
BeinertH.1983; Optical and EPR characterization of different species of active and inactive aconitase. Journal of Biological Chemistry 258:11106–11111
FinchP. W.,
WilsonR. E.,
BrownK.,
HicksonI. D.,
EmmersonP. T.1986a; Complete nucleotide sequence of the Escherichia coliptr gene encoding protease III. Nucleic Acids Research 19:7695–7703
FinchP. W.,
WilsonR. E.,
BrownK.,
HicksonI. D.,
TomkinsonA. E.,
EmmersonP. T.1986b; Complete nucleotide sequence of the Escherichia coli recC gene and the thyA–recC intergenic region. Nucleic Acids Research 14:4437–4451
GangloffS. P.,
MarguetD.,
LauquinG. J.-M.1990; Molecular cloning of the yeast mitochondrial aconitase gene (ACOI) and evidence of a synergistic regulation of expression by glucose plus glutamate. Molecular and Cellular Biology 10:3551–3561
GrayC. T.,
WimpennyJ. W. T.,
MossmanM. R.1966; Regulation of metabolism in facultative bacteria. II. Effects of aerobiosis and nutrition on the formation of Krebs cycle enzymes in Escherichia coli
. Biochimica et Biophysica Acta 117:33–41
KennedyM. C.,
BienertH.1983; The state of cluster SH and S2– of aconitase during cluster interconversions and removal. Journal of Biological Chemistry 263:8194–8198
KennedyM. C.,
EmptageM. H.,
DreyerJ.-L.,
BienertH.1983; The role of iron in the activation-inactivation of aconitase. Journal of Biological Chemistry 258:11098–11105
KennedyM. C.,
WerstM.,
TelserJ.,
EmptageM. H.,
BeinertH.,
HoffmanB. M.1987; Mode of substrate carboxyl binding to the [4Fe-4S]+ cluster of reduced aconitase as studied by 17O and 13C electron-nuclear double resonance spectroscopy. Proceedings of the National Academy of Sciences of the United States of America848854–8858
KoharaY.,
AkiyamaK.,
IsonoK.1987; The physical map of the whole E. coli chromosome : application of a new strategy for rapid analysis and sorting of a large genomic library. Cell 50:495–508
MasonP. J.,
WilliamsJ. G.1985; Hybridization analysis of DNA. Nucleic Acid Hybridization, a Practical Approach113–137HamesB. D.,
HigginsS. J.
Oxford: IRL Press;
RobbinsA. H.,
StoutC. D.1989b; Structure of activated aconitase: Formation of the [4Fe-4S] cluster in the crystal. Proceedings of the National Academy of Sciences of the United States of America863639–3643
RosenkrantzM. S.,
DingmanD. W.,
SonensheinA. L.1985; Bacillus subtilis citB gene is regulated synergistically by glucose and glutamine. Journal of Bacteriology 164:155–164
RydenL.,
OfverstedtL.,
BeinertH.,
EmptageH.,
KennedyM. C.1984; Molecular weight of beef heart aconitase and stoichiometry of the components of its iron-sulfur cluster. Journal of Biological Chemistry 259:3141–3144
ScholzeH.1983; Studies on aconitase species from Saccharomyces cerevisiae porcine and bovine heart, obtained by a modified isolation method. Biochemica et Biophysica Acta 746:133–137
SuzukiT.,
YamazakiO.,
NaraK.,
AkiyamaS.-I.,
NakaoY.,
FukudaH.1975; The aconitase of yeast: II. crystallization and general properties of yeast aconitase. Journal of Biochemistry 77:367–372
WerstM. M.,
KennedyM. C.,
BienertH.,
HoffmanB. M.1990a; 17O, 1H, and 2H electron double resonance characterization of solvent, substrate, and inhibitor binding to the [4Fe-4S]+ cluster of aconitase. Biochemistry 29:10526–10532
WerstM. M.,
KennedyM. C.,
BienertH.,
HousemanA. L. P.,
HoffmanB. M.1990b; Characterization of the [4Fe-4S] cluster at the active site of aconitase by 57Fe, 33S, and 14N electron double resonance spectroscopy. Biochemistry 29:10533–10540
WoodsS. A.,
SchwartzbachS. D.,
GuestJ. R.1988; Two biochemically distinct classes of fumarases in Escherichia coli
. Biochimica et Biophysica Acta 954:14–26
ZhengL.,
AndrewsM. A.,
HermodsonM. A.,
DixonJ. E.,
ZalkinH.1990; Cloning and structural characterization of porcine heart aconitase. Journal of Biological Chemistry 26:2814–2821