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The MSMEG_1586 of M. smegmatis Is a Penicillin-Interactive Enzyme That Can Potentially Hydrolyse Aztreonam and
Aditya Prasad Panda1, Satya Deo Pandey1,2, Diamond Jain1
1Department of Biosciences and Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, 721302, India.
Abstract:
Mycobacteria are intrinsically resistant to beta-lactams as they possess several putative penicillin-interactive enzymes (PIEs), some of those are with dual-activity, namely DD-carboxypeptidase and beta-lactamase. Here, with help of molecular approaches, we elucidated the nature of one such putative PIE, MSMEG_1586, in Mycobacterium smegmatis. The in vivo expression of the membrane-bound form of MSMEG_1586 enhanced the beta-lactam resistance of a beta-lactamase deleted host E. coli strain (AM1OC), particularly for aztreonam (eight-fold) and cephalosporins (8-16 fold). To understand the reason for such elevation of resistance, soluble-form of MSMEG_1586 (sMSMEG_1586) was created by removing signal peptides and partially eliminating the amphipathic helix, and finally, expressed and purified. The purified sMSMEG_1586 was active and manifested a strong penicillin-binding affinity as shown by its ability to bind to fluorescent penicillin (Bocillin-FL). Interestingly, the steady-state kinetics apparently confirmed the hydrolytic ability of sMSMEG_1586 towards cefotaxime and aztreonam where hydrolysing aztreonam is a unique and rare behaviour among the beta-lactamases. However, sMSMEG_1586 was devoid of exerting DD-carboxypeptidase like activity. Finally, in silico analysis of MSMEG_1586 revealed a special folding that resembles class C beta-lactamase, except for the absence of a characteristic R2 loop. Overall, MSMEG_1586 could be categorized as a cephalosporinase with the ability to hydrolyse aztreonam.
Insights
Mycobacterium smegmatis possesses a novel enzyme, MSMEG_1586, which enhances resistance to beta-lactam antibiotics like aztreonam and cephalosporins. This enzyme acts as a cephalosporinase, uniquely hydrolyzing aztreonam.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Mycobacteria exhibit intrinsic resistance to beta-lactam antibiotics.
- This resistance is often mediated by penicillin-interactive enzymes (PIEs), some with dual DD-carboxypeptidase and beta-lactamase activity.
Purpose of the Study:
- To elucidate the function of the putative PIE, MSMEG_1586, in Mycobacterium smegmatis.
- To characterize its role in beta-lactam resistance and its enzymatic properties.
Main Methods:
- Molecular approaches were used to study MSMEG_1586.
- In vivo expression in a beta-lactamase deleted E. coli strain (AM1OC).
- Purification and characterization of soluble MSMEG_1586 (sMSMEG_1586), including binding assays (Bocillin-FL) and kinetic analysis, alongside in silico structural analysis.
Main Results:
- Expression of membrane-bound MSMEG_1586 increased beta-lactam resistance in E. coli, notably against aztreonam (8-fold) and cephalosporins (8-16 fold).
- Purified sMSMEG_1586 showed strong penicillin-binding affinity and hydrolytic activity against cefotaxime and aztreonam.
- Aztreonam hydrolysis is a rare characteristic for beta-lactamases; sMSMEG_1586 lacked DD-carboxypeptidase activity.
- In silico analysis revealed a Class C beta-lactamase-like fold, lacking a typical R2 loop.
Conclusions:
- MSMEG_1586 functions as a cephalosporinase with a unique ability to hydrolyze aztreonam.
- This enzyme contributes significantly to the beta-lactam resistance of Mycobacterium smegmatis.
- The structural features of MSMEG_1586 differentiate it from typical beta-lactamases.
