Does Escherichia coli possess a second citrate synthase gene?
Escherichia coli possesses a hexameric citrate synthase that exhibits allosteric kinetics and regulatory sensitivity, and for which the gene (gltA) has previously been cloned and sequenced. A citrate-synthase-deficient strain of E. coli (K114) has been mutated to generate a revertant (K114r4) that produces a dimeric citrate synthase with altered kinetic and regulatory properties. On cloning and sequencing the gltA gene from both K114 and K114r4, a single mutation was found that caused the replacement of Asp362 with Asn. Asp362 has been previously shown to be a catalytically essential residue in E. coli citrate synthase, and we demonstrate that the hexameric enzyme produced on expression of the gltA gene from K114 and K114r4 is inactive. The dimeric citrate synthase from K114r4 has been purified and shown to be immunologically distinct from the wild-type hexameric enzyme. Determination of its N-terminal amino acid sequence demonstrates that the mutant citrate synthase is encoded by a gene distinct from the E. coli gltA gene. The N-terminal sequence is compared with those of other eukaryotic, eubacterial and archaebacterial citrate synthases.
Department of Biochemistry, University of Bath, England.
A J
AJ
Patton
D W
DW
Hough
P
P
Towner
M J
MJ
Danson
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides
D009838:Oligodeoxyribonucleotides; D002950:Citrate (si)-Synthase