Gabaculine-resistant glutamate 1-semialdehyde aminotransferase of Synechococcus. Deletion of a tripeptide close to the NH2 terminus and internal amino acid substitution.
Glutamate 1-semialdehyde aminotransferase (GSA-AT) is the last enzyme in the C5 pathway converting glutamate into the tetrapyrrole precursor delta-aminolevulinate in plants, algae, and several bacteria. Sequence analysis of the genes encoding GSA-AT in barley, Synechococcus, and Escherichia coli revealed 50-70% similarity in the primary structures of the proteins. The enzyme is inhibited rapidly by gabaculine when added in approximately stoichiometric amounts with the enzyme. A gabaculine-tolerant Synechococcus strain, GR6, was found to produce a GSA-AT less sensitive to the inhibitor. Accordingly, the mutant gene was isolated and sequenced. In comparison with the wild-type gene it contains a deletion of nine nucleotides (position 12-20) and a guanine to adenine substitution (position 743). This resulted in the loss of the amino acids serine, proline, and phenylalanine (position 5-7) close to the NH2 terminus of the enzyme and an exchange of Met-248 for isoleucine in the middle of the polypeptide chain. Wild-type and mutant GSA-AT were expressed in E. coli and purified close to homogeneity. Although the specific activity of the mutant GSA-AT was only one-fifth of the wild type, it displayed a 100-fold increased resistance to gabaculine. Peaks in the absorption spectrum of the purified recombinant GSA-ATs at 335 and 417 nm are typical of a transaminase containing a B6 cofactor. Incubation with substrate and with inhibitor induced spectral changes characteristic of other gabaculine-sensitive, B6-requiring enzymes.
Grimm
B
B
Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, Copenhagen, Denmark.
Smith
A J
AJ
Kannangara
C G
CG
Smith
M
M
D000596:Amino Acids / Q000235:genetics; D001483:Base Sequence; D002848:Chromatography, DEAE-Cellulose; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D005822:Genetic Vectors; D019751:Intramolecular Transferases; D007535:Isomerases / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D011995:Recombination, Genetic
D000596:Amino Acids; D003509:Cyclohexanecarboxylic Acids; D004269:DNA, Bacterial; C012987:gabaculine; D007535:Isomerases; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase