The Molecular Structure and Catalytic Mechanism of a Novel Carboxyl Peptidase from Scytalidium lignicolum

• Published in: Proceedings of the National Academy of Sciences - PNAS 2004-03, Vol.101 (10), p.3364-3369
• Main Authors: Fujinaga, Masao, Cherney, Maia M., Oyama, Hiroshi, Oda, Kohei, Michael N. G. James, Davies, David R.
• Format: Article
• Language: English
• Subjects: Active sites; Amino Acid Sequence; Angiotensin II - metabolism; Ascomycota - enzymology; Ascomycota - genetics; Aspartic Acid Endopeptidases - chemistry; Aspartic Acid Endopeptidases - genetics; Aspartic Acid Endopeptidases - metabolism; Atoms; Biochemistry; Biological Sciences; Carboxyl compounds; Carboxylates; Catalysis; Catalytic Domain; Crystallography, X-Ray; Crystals; Electron density; Enzymes; Fungi; Hydrogen Bonding; Hydrogen bonds; Hydrolysis; Models, Molecular; Molecular Sequence Data; Molecular structure; Molecules; Nitrogen; Peptides; Protein Conformation; Scytalidium lignicolum; Sequence Homology, Amino Acid; Static Electricity; Terminology as Topic; Water
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0400246101

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 ( Rfree=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 β-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 carbonyl-carbon 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.