Abstract
The aminopeptidase from Streptomyces griseus (SGAP) has been cloned and expressed in Escherichia coli. By growing the cells in the presence of 1 M sorbitol at 18°C, the protein was obtained in a soluble and active form. The amino acid sequence of the recombinant SGAP contained four amino acids differing from the previously published sequence. Re-sequencing of the native protein indicated that asparagines 70 and 184 are in fact aspartic acids as in the recombinant protein. Based on the crystal structure of SGAP, Glu131 and Tyr246 were proposed to be the catalytic residues. Replacements of Glu131 resulted in loss of activity of 4-5 orders of magnitude, consistent with Glu131 acting as the general base residue. Mutations in Tyr246 resulted in about 100-fold reduction of activity, suggesting that this residue is involved in the stabilization of the transition state intermediate.
| Original language | English |
|---|---|
| Pages (from-to) | 192-196 |
| Number of pages | 5 |
| Journal | FEBS Letters |
| Volume | 571 |
| Issue number | 1-3 |
| DOIs | |
| State | Published - 30 Jul 2004 |
Bibliographical note
Funding Information:This study was supported by the Otto Meyerhof Center for Biotechnology at the Technion, established by the Minerva Foundation (Munich, Germany) and the Israel Science Foundation (ISF). V.R. was supported by a Marie Curie Fellowship of the European Commission and by a Lady Davis Fellowship from the Hebrew University of Jerusalem, Israel.
Keywords
- Aminopeptidase
- Catalytic mechanism
- Leu-pNA, Leucine-para-nitroanilide
- SGAP, Streptomyces griseus aminopeptidase
- Soluble expression
- Sorbitol
- Streptomyces griseus