A CLN2-Related and Thermostable Serine-Carboxyl Proteinase, Kumamolysin: Cloning, Expression, and Identification of Catalytic Serine Residue

• Published in: Journal of biochemistry (Tokyo) Vol. 131; no. 5; pp. 757 - 765
• Main Authors: Oyama, Hiroshi, Hamada, Takatoshi, Ogasawara, Shin, Uchida, Kenichi, Murao, Sawao, Beyer, Bret B., Dunn, Ben M., Oda, Kohei
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.05.2002
• Subjects: Amino Acid Sequence; Aspartic Acid Endopeptidases - antagonists & inhibitors; Aspartic Acid Endopeptidases - chemistry; Aspartic Acid Endopeptidases - genetics; Aspartic Acid Endopeptidases - metabolism; Base Sequence; Blotting, Western; Catalytic Domain; catalytric triad; Cloning, Molecular; Enzyme Stability; Escherichia coli - enzymology; Hot Temperature; Kinetics; kumamoysin; Molecular Sequence Data; Mutation; pepstatin-insensitive carboxyl proteinase; Protease Inhibitors - pharmacology; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Restriction Mapping; Sequence Homology; Serine - metabolism; serine-carboxyl proteinase; Structure-Activity Relationship; thermostable proteinase
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a003162

The gene encoding kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase, was cloned and expressed, (i) Kumamolysin was synthesized as a large precursor consisting of two regions: amino-terminal prepro (188 amino acids) and mature proteins (384 amino acids), (ii) The deduced amino acid sequence of the mature region exhibited high similarity to those of such bacterial pepstatin-insensitive enzymes as Pseudomonas carboxyl proteinase (PSCP; EC 3.4.23.37, identity = 37%), Xanthomonas carboxyl proteinase (XCP; EC 3.4.23.33, identity = 36%), and human CLN2 gene product (identity = 36%), which is related to a fatal neurodegenerative disease, (iii) The presumed catalytic triad, Glu78, Asp82, Ser278 [three-dimensional structure of PSCP: Wlodawer, A. et al. (2001) Nature Struct BioL, 8, 442–446], was found to be conserved in the amino acid sequence of kumamolysin. (iv) Kumamolysin was inactivated by such aldehyde-type inhibitors as Ac-Ile-Pro-Phe-CHO (Ki = 0.7 ± 0.14 μM). In PSCP, it has been clarified that these inhibitors form a hemiacetal linkage with the catalytic serine residue and inactivate the enzyme, (v) Mutational analysis of the Ser278 residue revealed that the mutant lost both auto-processing activity and proteolytic activity. These results strongly suggest that kumamolysin has a unique catalytic triad consisting of Glu78, Asp82, and Ser278 residues, as previously observed for PSCP.