Cloning and sequence analysis of cDNA encoding endopolygalacturonase I from Stereum purpureum.
Endopolygalacturonase (endoPG) I was obtained from Stereum purpureum by an improved easier purification procedure. It was found that EndoPG I consisted of three glycosilated proteins with the same isoelectric point and different molecular masses, 42, 45, and 48 kDa, respectively. However, the enzymatic deglycosilation product of endoPG I gave a single band at the position corresponding to 39kDa on SDS-PAGE. Furthermore, the N-terminal amino acid sequences of three endoPGs were identical one another up to 20 residues. A cDNA library was constructed and positive cDNA clones encoding endoPG I were isolated by using antibody raised against the purified endoPG I. Nucleotide sequence analysis of the cDNA disclosed a 1212-bp open reading frame that encoded 403 amino acid residues. The N-terminal amino acid sequence (residues 1-20) of endoPG I coincided with the deduced amino acid sequence starting from the 25th residue. Therefore, the sequence of the first 24 residues represented a signal peptide and the remaining sequence, consisting of 379 residues, was the mature protein with molecular mass of 39.1 kDa. The deduced sequence of endoPG I showed 30-45% similarity in comparison with those of bacterial and fungal endoPGs, and the sequence of putative active site residues reported for the endoPGs was highly conserved in the sequence of endoPG I.
Miyairi
K
K
Department of Bioresources Science, Faculty of Agriculture, Hirosaki University, Aomori, Japan.
Senda
M
M
Watanabe
M
M
Hasui
Y
Y
Okuno
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D001487:Basidiomycota / Q000201:enzymology*; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011096:Polygalacturonase / Q000235:genetics*; D014176:Protein Biosynthesis; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011096:Polygalacturonase