|
type |
Journal Article |
authors |
Grimm, B.; Smith, A. J.; Kannangara, C. G.; Smith, M. |
title |
Gabaculine-resistant glutamate 1-semialdehyde aminotransferase of Synechococcus. Deletion of a tripeptide close to the NH2 terminus and internal amino acid substitution |
journal |
J Biol Chem |
Activity |
5.4.3.8 |
Family |
5.4.3.8 |
ui |
GrgsaSD |
year |
(1991) |
volume |
266 |
number |
19 |
pages |
12495-501 |
| |
keywords |
Amino Acids/genetics |
abstract |
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. |
last changed |
2008/05/21 16:26 |
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