Cloning and Expression of an Isovaleryl Pepstatin-Insensitive Carboxyl Proteinase Gene from Xanthomonas sp. T-22

Abstract

Xanthomonas carboxyl proteinase (XCP), isolated from Xanthomonas sp. T-22, is the second example of the unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to the classical aspartic proteinase inhibitor. The gene coding for XCP was cloned, sequenced, and expressed in Escherichia coli. The XCP gene contains an open reading frame of 2,481 base pairs encoding a protein of 827 amino acid residues with a M(r) of 83,677. The XCP was synthesized as a large precursor consisting of three regions: NH2-terminal prepro (N-Prepro) (237 amino acid residues); mature XCP (398 a.a.residues); and COOH-terminal pro (C-Pro) (192 a.a. residues). The N-Prepro and mature XCP regions had no sequence similarity to any other proteins reported so far, except the carboxyl proteinase from Pseudomonas sp. 101 [Oda, K., Takahashi, T., Tokuda, Y., Shibano, Y., and Takahashi, S. (1994) J. Biol. Chem. 269, 26518-26524]. The C-Pro region showed high similarity to COOH-terminal regions of other microbial proteinase precursors. E. coli carrying a plasmid containing the cloned wild-type XCP gene produced an 84-kDa protein. This protein was processed into a mature, active form under acidic conditions. This process was completely blocked by tyrostatin, an XCP-specific inhibitor from Kitasatosporia sp. 55, indicating an autocatalytic processing. The purified recombinant XCP had the same characteristics as authentic XCP except for the NH2-terminal amino acid sequence. When the mutant XCP gene truncated in the C-Pro region was expressed in E. coli, an expected 64-kDa protein was detected in the cells, and also processed into the 42-kDa active form under the acidic conditions. Thus, the C-Pro region was not essential for the formation of active mature XCP.

Molecular Analysis of the Gene Encoding a Novel Chitin-Binding Protease from Alteromonas sp. Strain O-7 and Its Role in the Chitinolytic System

“…However, this insertion sequence was missing in AprIV. PkdD, which was first identified in human polycystin-1 (6), is also found in bacterial collagenases (16), proteases (19), cellulases (1), and chitinases (21). This domain has a ␤-sandwich fold, and the sequence, WDFGDG, is highly conserved in the domain (4).…”

The Human CLN2 Protein/Tripeptidyl-Peptidase I Is a Serine Protease That Autoactivates at Acidic pH

“…The CLN2 gene product was first identified as a normally abundant 46-kDa mannose 6-phosphorylated glycoprotein that was absent in brain autopsy specimens from LINCL patients, leading to the molecular characterization of the disease gene (2). The encoded protein has significant sequence similarities to two previously characterized bacterial endoproteases from Xanthomonas and Pseudomonas (3,4). These prokaryotic enzymes have been named bacterial pepstatin-insensitive carboxyl peptidases (BPICPs) based on a series of studies that suggest that, like classic aspartyl proteases, their catalytic mechanism involves a pair of amino acids with carboxyl side chains that catalyze peptide bond hydrolysis at acidic pH but, unlike the classic aspartyl proteases, they are not inhibited by pepstatin.…”

Mechanistic Insight into the Function of the C-terminal PKD Domain of the Collagenolytic Serine Protease Deseasin MCP-01 from Deep Sea Pseudoalteromonas sp. SM9913

“…In addition to cell surface proteins, the PKD domain is found in many biopolymer hydrolases, such as chitinases (4,5), celluloses (6), and proteases (7)(8)(9), suggesting that it may play an important role in biopolymer degradation. The structures of three PKD domains have been solved, which show that, though their sequences are different, they all adopt a ␤-helix fold and a conserved sequence area with two Trp residues in the hydrophobic core (2,3).…”