Identification of a glutamine residue essential for catalytic activity of aspergilloglutamic peptidase by site-directed mutagenesis.
Aspergilloglutamic peptidase (AGP, formerly called aspergillopepsin II) from Aspergillus niger var. macrosporus is a unique acid protease recently classified to the peptidase family G1. Our previous study using site-directed mutagenesis on the glutamic and aspartic acid residues of AGP conserved among the G1 family suggested that Glu219 and Asp123 (numbering in the preproform) are important for catalytic activity. However, the Asn mutant of Asp123 retained weak but significant activity and therefore it was unclear whether it is an active site residue. In this study, we performed site-directed mutagenesis on all the other hydrophilic residues including Gln, Asn, Ser, Thr, and Tyr, conserved in this family to screen other residues that might be essential for catalytic function, and found that mutations of only Gln133 resulted in almost complete loss of enzymatic activity without change in the native conformation of the enzyme. Meanwhile, the 3D structure of scytalidoglutamic peptidase, a homologue from Scytalidium lignicolum, has been reported, indicating that Glu136 and Gln53 (the counterparts of Glu219 and Gln133 in AGP) form a catalytic dyad. Therefore, the results obtained in this and our previous studies provide with complementary evidence for the definitive conclusion on the catalytic function of the Glu/Gln dyad in glutamic peptidases.
Yabuki
Yutaka
Y
Laboratory of Molecular Biochemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Kubota
Keiko
K
Kojima
Masaki
M
Inoue
Hideshi
H
Takahashi
Kenji
K
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001234:Aspergillus niger / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D017931:DNA Primers; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005973:Glutamine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D005656:Fungal Proteins; D011994:Recombinant Proteins; D005973:Glutamine; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C022442:aspergillopepsin II