Abstract
Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.
Highlights
Proteolytic enzymes catalyze the cleavage of peptide bonds in proteins and are divided into several major classes based on their mechanism of catalysis [1,2]
We chose a set of proteases with known 3D structures and active site residues from each of the seven major classes in the MEROPS database (Table 1) [3]
To expand our previous work on alkaline phosphatases (AP), we investigated the proteolytic activity of a cold-active Vibrio AP (VAP) [18] on four substrates: benzoyl-Arg-pNA, Z-GlyProArg-pNA, succinyl-AlaAlaAla-pNA, and succinyl-AlaAlaProPhe-pNA
Summary
Proteolytic enzymes catalyze the cleavage of peptide bonds in proteins and are divided into several major classes based on their mechanism of catalysis [1,2]. The distinct types of proteases categorized in the MEROPS database were used to generate a search module that could be used to query any protein with known 3D structure for the presence of a promiscuous proteolytic activity. This search module identified a serine protease scaffold in PI-PLC from Bacillus cereus, which was validated by in vitro experiments. A similar computational approach can be adopted for other enzymatic functions to extend protein families based on the spatial and electrostatic congruence of active site residues: relationships that often escape detection by sequence alignment or global structure alignment methods
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
