Identification of the catalytic residues in the double-zinc aminopeptidase from Streptomyces griseus

• Published in: FEBS letters 2004-07, Vol.571 (1), p.192-196
• Main Authors: Fundoiano-Hershcovitz, Yifat, Rabinovitch, Larisa, Langut, Yael, Reiland, Vera, Shoham, Gil, Shoham, Yuval
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Aminopeptidase; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; Catalytic Domain; Catalytic mechanism; DNA Primers; Escherichia coli; Kinetics; Leu-pNA, Leucine-para-nitroanilide; Leucyl Aminopeptidase - chemistry; Leucyl Aminopeptidase - genetics; Leucyl Aminopeptidase - metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Restriction Mapping; SGAP, Streptomyces griseus aminopeptidase; Soluble expression; Sorbitol; Streptomyces griseus; Streptomyces griseus - enzymology
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2004.07.001

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.