Abstract
The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production.
Highlights
Tuberculosis (TB), which is one of the main causes of death from infectious diseases, accounted for 1.4 million deaths in 2011 (World Heath Organisation, Global Tuberculosis report 2012, http://www.who.int/tb/publications/global_report/en/index. html)
Despite the high level of homology known to exist between their amino acid sequences, the biochemical data obtained on Cfp21 and Cut4 indicated that these two proteins have distinct substrate specificities [7] showing that Cfp21 is a lipase and Cut4 is a strict phospholipase
In silico molecular docking studies showed that these three regions are close to the active site and able to interact with the transition states of a TAG glyceroltricaprate, and confirmed the choice of these areas in the subsequent site-directed mutagenesis analyses
Summary
Tuberculosis (TB), which is one of the main causes of death from infectious diseases, accounted for 1.4 million deaths in 2011 (World Heath Organisation, Global Tuberculosis report 2012, http://www.who.int/tb/publications/global_report/en/index. html). More than 2 billion people are infected by Mycobacterium tuberculosis, the etiologic agent responsible for the disease, and 8 million people develop active TB every year. M. tuberculosis can survive in the host inside structures called granulomas that enable the bacteria to escape the host’s immune system [2]. The mechanism whereby lipids are transferred from the host to the pathogen has not yet been completely elucidated, but recent results have suggested that lipolytic enzymes may play a major role in the life cycle of the mycobacteria [7,8,9]. Further characterization of the M. tuberculosis lipolytic enzymes should lead to the development of new strategies for treating tuberculosis. Many lipolytic enzymes have been identified in the genome sequence of
Sign-in/Register to view highlights & summary 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.
