Structural and biochemical characterization of the Clostridium perfringens autolysin catalytic domain

Abstract

Bacterial autolysins can partially hydrolyze cell wall peptidoglycans into small sections to regulate cell separation/division and the growth phase. Clostridium perfringens autolysin (Acp) has an N-terminal cell wall-binding domain and a C-terminal catalytic domain with glucosaminidase activity that belongs to the glycoside hydrolase 73 family. Here, we determined the X-ray structure of the Acp catalytic domain (AcpCD) at 1.76 A resolution. AcpCD has a unique crescent-shaped structure, forming a deep groove for substratebinding at the center of the protein. The modeling study of the enzyme/substrate complex demonstrated that the length of the substrate-binding groove is closely related to the glucosaminidase activity. Mutagenesis analysis showed that AcpCD likely adopts a neighboring-group mechanism for the catalytic reaction.

Domain sliding of two Staphylococcus aureus N-acetylglucosaminidases enables their substrate-binding prior to its catalysis

“…The mutation of the tyrosine residue Y214 in the YATD region to alanine severely affected enzyme activity, whereas mutation of Y214 to phenylalanine completely restored the substrate degrading ability. Similar observations were previously established for AtlE 17 , as well as for four other GH73 enzymes 24,25,28,32,33 , demonstrating that an aromatic residue is required at this position. The T216A and D217N mutants exhibited a marked decrease in activity.…”

“…All produced AtlA-gl mutants were correctly folded as confirmed by CD spectra. Mutation of the catalytic glutamate E116 to alanine rendered enzymes inactive, as has been shown previously for several enzymes in this family, including AtlE 17 , AtlWM 24 , AcmA 25 , LytB 26,27 , SpFlgJ 28 , StFlgJ 29 , Auto 30 , TM0633 31 , and AcpCD 32 . The mutation of the tyrosine residue Y214 in the YATD region to alanine severely affected enzyme activity, whereas mutation of Y214 to phenylalanine completely restored the substrate degrading ability.…”

Catalytic diversity and cell wall binding repeats in the phage‐encoded endolysins

“…A similar groove is present in all four GH73 domain enzymes (Fig. A), and molecular modeling supports similar binding modes for other bacterial autolysins (Bai et al ., ; Tamai et al ., ). Our analysis here indicates that a similar binding groove, due to both sequence and structure conservation, also exists in the endolysin domain PlyC GyH (Figs A).…”

Structural Investigations on the SH3b Domains of Clostridium perfringens Autolysin through NMR Spectroscopy and Structure Simulation Enlighten the Cell Wall Binding Function

“…C. perfringens autolysin (CpAcp, UniProt ID: Q8XL11) encoded by gene CPE1231 is a peptidoglycan hydrolase expressed at growth phase and sporophore formative phase, and is associated with cell separation, division, and growth [ 2 , 7 ]. CpAcp has 1129 amino-acid residues and consists of a signal peptide, a CWB module containing ten tandem bacterial Src homology 3 (SH3b) domains, and a catalytic domain belonging to the N-acetylglucosaminidase family ( Figure 1 ) [ 7 , 8 ]. The crystal structure of CpAcp catalytic domain has been determined to adopt a crescent-shaped fold with a deep groove at the center for substrate binding [ 8 ].…”

“…CpAcp has 1129 amino-acid residues and consists of a signal peptide, a CWB module containing ten tandem bacterial Src homology 3 (SH3b) domains, and a catalytic domain belonging to the N-acetylglucosaminidase family ( Figure 1 ) [ 7 , 8 ]. The crystal structure of CpAcp catalytic domain has been determined to adopt a crescent-shaped fold with a deep groove at the center for substrate binding [ 8 ]. However, the structures of the ten SH3b domains of CpAcp have not been reported so far.…”