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

The molecular structure and catalytic mechanism of a novel carboxyl peptidase from Scytalidium lignicolum Masao Fujinaga*, Maia M. Cherney*, Hiroshi Oyama†, Kohei Oda†, and Michael N. G. James*‡ *Canadian Institutes of Health Research Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7; and †Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Sakyo-ku, Kyoto 6068585, Japan Communicated by David R. Davies, National Institutes of Health, Bethesda, MD, January 22, 2004 (received for review December 1, 2003) The molecular structure of the pepstatin-insensitive carboxyl pepti- intimately involved in the catalytic function. SCP-B is not inhibited dase from Scytalidium lignicolum, formerly known as scytalidopepsin by pepstatin, acetyl-pepstatin, nor by diazoacetyl-DL-norleucine B, was solved by multiple isomorphous replacement phasing methods methyl ester, but it is inhibited by 1,2-epoxy-3-(p-nitrophenoxy)pro- ,at 2.1-Å resolution. pane. SCP-B cleaves the oxidized insulin B chain at Tyr-26–Thr-27 (0.246 ؍ and refined to an R factor of 0.230 (Rfree In addition to the structure of the unbound peptidase, the structure Phe-24–Phe-25, and other positions after Tyr and Glu. It has also of a product complex of cleaved angiotensin II bound in the active site been shown to cleave the His-6–Pro-7 bond of angiotensin II and, of the enzyme was also determined. We propose the name scytali- to a lesser extent, the bond between Tyr-4 and Ile-5 of this docarboxyl peptidase B (SCP-B) for this enzyme. On the basis of octapeptide (15). conserved, catalytic residues identified at the active site, we suggest Several studies have been carried out to identify the catalytic the name Eqolisin for the enzyme family. The previously uninvesti- residues of the eqolisins. Tsuru and colleagues (16, 17) reported gated SCP-B fold is that of a ␤-sandwich; each sheet has seven that Glu-53 and Asp-98, residues in SCP-B, were candidates for its antiparallel strands. A tripeptide product, Ala-Ile-His, bound in the catalytic residues. The nucleotide sequence of the SCP-B gene, active site of SCP-B has allowed for identification of the catalytic elucidated after these studies, however, revealed that Glu-53 was in residues and the residues in subsites S1, S2, and S3, which are fact a glutamine residue (18). Furthermore, it turned out that important for substrate binding. The most likely hydrolytic mecha- Asp-98 is not conserved in any of the other members of this nism involves nucleophilic attack of a general base (Glu-136)-acti- peptidase family. Takahashi and colleagues (19) reported from vated water (OH؊)onthesi-face of the scissile peptide carbonyl- mutational data that Glu-219 and Asp-123 (sequence numbers in carbon atom to form a tetrahedral intermediate. Electrophilic the mature enzyme, Glu-149 and Asp-53) in AnCPA were involved assistance and oxyanion stabilization is provided by the side-chain in the catalytic function. These residues are conserved in the SCP-B amide of Gln-53. Protonation of the leaving-group nitrogen is accom- molecule as Glu-136 and Asp-43, respectively. Thus, without struc- plished by the general acid function of the protonated carboxyl group tural data, the catalytic mechanism for this unique family of of Glu-136. peptidases is still shrouded in mystery. We have solved the crystal structures of SCP-B in the unbound epstatin-insensitive carboxyl peptidases (1–8) originally found form and in a form complexed with hydrolytic products of angio- Pby Murao and Oda can be classified into two groups, bacterial tensin II. On the basis of these 3D structures and of sequence and and fungal carboxyl peptidases. The 3D structures of the two biochemical data, it was determined that SCP-B has a catalytic dyad members of the bacterial carboxyl peptidases have recently been consisting of residues Gln-53 and Glu-136. These two residues and determined (9, 10). From the tertiary folds, it was clear that these the surrounding segments of polypeptide chain are highly con- enzymes are members of the subtilisin family. Each enzyme has a served in all fungal pepstatin-insensitive carboxyl peptidases of the glutamate residue acting as the general base instead of the histidine eqolisin family (Fig. 1). From the results derived here, the eqolisins should occupy a more distinct position in the overall classification present in the subtilisins. The environments of the carboxylates of peptidases (20). contribute to the low pH optima of these enzymes. The fungal pepstatin-insensitive carboxyl peptidases (5) are Methods represented by several enzymes, among them the enzyme that is the Crystallization and Data Collection. Lyophilized SCP-B was dissolved subject of this article. Formerly, this enzyme has been called in water at 20 mg͞ml. Crystals were grown at room temperature by Scytalidopepsin-B because of its low pH optimum and preponder- the hanging-drop method from 42% saturated ammonium sulfate͞ ance of acidic residues (8). It is isolated from Scytalidium lignicolum 0.1 M sodium acetate buffer (pH 4.0)͞10% (vol/vol) ethylene ATCC24568. We have chosen to rename this enzyme scytalido- glycol. Before data collection, crystals of SCP-B were transferred carboxyl peptidase-B (SCP-B) to reflect its fungal source and for several seconds into a cryosolution containing 30% glycerol, active-site carboxyl groups. SCP-B is synthesized as a precursor 45% saturated ammonium sulfate, and 0.1 M sodium acetate (pH consisting of two regions: an amino-terminal preprosegment of 54 4.0) then frozen in the cryostream. Heavy-atom derivative crystals amino acid residues and a mature enzyme consisting of 206 amino were obtained by soaking native crystals in 1.5 mM mersalyl for 4 h acid residues (11). The mature enzyme has no sequence similarity or in 3 mM uranyl acetate overnight before freezing and data to the well known pepsin-like or retroviral aspartic peptidases, but collection. For soaking a potential substrate into the crystals, a drop it has significant similarity to the other fungal pepstatin-insensitive consisting of 3 ␮l of 10 mM angiotensin II in water and 3 ␮lofthe carboxyl peptidases (Fig. 1). crystallization solution was equilibrated for several hours and an SCP-B and the other carboxyl peptidases described here have SCP-B crystal was soaked in it overnight. The diffraction data were been classified in family A4 of the aspartic endopeptidases in the collected the next day from a flash-frozen crystal. MEROPS (http:͞͞merops.sanger.ac.uk). database. On the basis of our structural results, however, we prefer to name this family the Eqolisins (pronounced ‘‘echo’’-lisin) derived from the active-site Abbreviations: SCP-B, scytalidocarboxyl peptidase B; AnCPA, Aspergillus niger carboxyl residues, glutamic acid (E) and glutamine (Q). We suggest that the peptidase A. eqolisins constitute a new peptidase family that is not part of the Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, aspartic peptidases. www.pdb.org (PDB ID codes 1S2B and 1S2K). SCP-B works best in acidic conditions, having an optimal pH of ‡To whom correspondence should be addressed. E-mail: [email protected]. 2.0 with casein as substrate, suggesting that carboxyl groups are © 2004 by The National Academy of Sciences of the USA 3364–3369 ͉ PNAS ͉ March 9, 2004 ͉ vol. 101 ͉ no. 10 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400246101 Downloaded by guest on September 29, 2021 All data except for the native set were collected on a Rigaku RAXIS-IVϩϩ area detector with CuK␣ radiation generated by a Rigaku RU-H3R rotating anode generator (Table 1). The native data were collected on beam line 9-1 at the Stanford Synchrotron Radiation Laboratory. All data were processed with the HKL suite of programs (21). Structure Determination and Refinement. The structure was solved by the multiple isomorphous replacement method from data collected from the mersalyl and uranyl heavy-atom derivatives by using the program SOLVE (22). SOLVE assigned one unique heavy-atom site for each of the derivatives. The phases derived from the multiple isomorphous replacement procedure resulted in a figure- of-merit (FOM) of 0.35. Subsequently, density modification carried out by the program RESOLVE (23, 24) increased the FOM to 0.68. Examination of the electron density computed from the phase set at this stage showed clearly interpretable molecular features. Thus, these phases and the corresponding electron density map were used for automated chain tracing by the program WARPNTRACE (25). The program was able to fit 168 residues in nine chain segments. Substitution of the correct sequence and additional model building was done manually with XTALVIEW (26). The program CNS (27) was used for refinement of both the native and product-bound structures (Table 1). The refined coordinates have been deposited in the Protein Data Bank with ID codes 1S2B and 1S2K. The crystal quality of SCP-B was variable. Despite the relatively high resolution recorded for the native data set, the quality of the ͞␴ Fig. 1. CLUSTALW multiple-sequence alignment of the eqolisins. The sequence data in the high-resolution shells was poor. Overall the mean I (I) numbering for each mature enzyme is indicated. The enzymes in this align- is 8.1; it is much worse (1.3) for the data in the range from 2.1 Å ment are SCP-B (11), Aspergillus niger carboxyl peptidase A (AnCPA) (12), to 1.9 Å. The value of Rmerge is also large for the same range of Sclerotinia sclerotiorum carboxyl peptidase (SSCP) (13), Cryphonectria para- data; overall, the data are only 89% complete. For these reasons, sitica peptidases B and C (EAPB and EAPC) (14), and Talaromyces emersonii the data were truncated to 2.1 Å for use in the refinement.

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