The molecular structure and catalytic mechanism of a novel carboxyl peptidase from Scytalidium lignicolum

Abstract

The molecular structure of the pepstatin-insensitive carboxyl peptidase from Scytalidium lignicolum, formerly known as scytalidopepsin B, was solved by multiple isomorphous replacement phasing methods and refined to an R factor of 0.230 (R free ‫؍‬ 0.246) at 2.1-Å resolution. In addition to the structure of the unbound peptidase, the structure of a product complex of cleaved angiotensin II bound in the active site of the enzyme was also determined. We propose the name scytalidocarboxyl peptidase B (SCP-B) for this enzyme. On the basis of conserved, catalytic residues identified at the active site, we suggest the name Eqolisin for the enzyme family. The previously uninvestigated SCP-B fold is that of a ␤-sandwich; each sheet has seven antiparallel strands. A tripeptide product, Ala-Ile-His, bound in the active site of SCP-B has allowed for identification of the catalytic residues and the residues in subsites S1, S2, and S3, which are important for substrate binding. The most likely hydrolytic mechanism involves nucleophilic attack of a general base (Glu-136)-activated water (OH ؊ ) on the si-face of the scissile peptide carbonylcarbon atom to form a tetrahedral intermediate. Electrophilic assistance and oxyanion stabilization is provided by the side-chain amide of Gln-53. Protonation of the leaving-group nitrogen is accomplished by the general acid function of the protonated carboxyl group of Glu-136. P epstatin-insensitive carboxyl peptidases (1-8) originally found by Murao and Oda can be classified into two groups, bacterial and fungal carboxyl peptidases. The 3D structures of the two members of the bacterial carboxyl peptidases have recently been determined (9, 10). From the tertiary folds, it was clear that these enzymes are members of the subtilisin family. Each enzyme has a glutamate residue acting as the general base instead of the histidine present in the subtilisins. The environments of the carboxylates contribute to the low pH optima of these enzymes.The fungal pepstatin-insensitive carboxyl peptidases (5) are represented by several enzymes, among them the enzyme that is the subject of this article. Formerly, this enzyme has been called Scytalidopepsin-B because of its low pH optimum and preponderance of acidic residues (8). It is isolated from Scytalidium lignicolum ATCC24568. We have chosen to rename this enzyme scytalidocarboxyl peptidase-B (SCP-B) to reflect its fungal source and active-site carboxyl groups. SCP-B is synthesized as a precursor consisting of two regions: an amino-terminal preprosegment of 54 amino acid residues and a mature enzyme consisting of 206 amino acid residues (11). The mature enzyme has no sequence similarity to the well known pepsin-like or retroviral aspartic peptidases, but it has significant similarity to the other fungal pepstatin-insensitive carboxyl peptidases (Fig. 1).SCP-B and the other carboxyl peptidases described here have been classified in family A4 of the aspartic endopeptidases in the MEROPS (http:͞͞merops.sanger.ac.uk). database. On the basis of our structur...

Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

“…The mature enzyme has significant sequence similarity to the other fungal carboxyl peptidases, Aspergillus niger carboxyl peptidase (ANCP), Sclerotinia sclerotiorum carboxyl peptidase, Cryphonectria parasitica peptidases B and C, and Talaromyces emersonii carboxyl peptidase [9].…”

“…It showed about 20% of the activity of that of the control. It has been clarified based on the structural analysis that the Asp43 residue was buried in the core of the SGP molecule and is essential to maintain the structure; it is not one of the catalytic residues [9]. The truncated form of the precursor in this case may be caused by its susceptibility to endogenous E. coli proteinases.…”

“…These residues are conserved in all of the enzymes described above. Glu136 and Gln53 were identified as the presumed catalytic residues by structural analysis [9]. Asp43 which corresponds to the Asp53 in ANCP, had been suggested as one of the catalytic residues by site-directed mutagenesis of ANCP [16].…”

“…In 2003, we solved the three-dimensional structure of SGP and the results were published in 2004 [9]. The SGP molecule is a b-sandwich structure composed of two 7-stranded antiparallel ß-sheets.…”

“…Protonation of the leavinggroup nitrogen is accomplished by the general acid function of the protonated carboxy group of Glu136. Based on these unique features, SGP has now been established as the sixth family of peptidases, the glutamic peptidase (G1) family in the MER-OPS database [8,9]. This family has been named the eqolisins.…”