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A Single Substitution of Valine to Glutamic Acid in the Omega-Like Loop Introduces a Penicillinase-Like Activity in

Aditya Prasad Panda1, Debasmita Chatterjee1, Anik Roy1

  • 1Department of Bioscience and Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, PIN-721302, West Bengal, India.

Current Microbiology
|August 10, 2026
PubMed

Insights

Mycobacterium smegmatis enzyme MSMEG_6194 functions as a DD-carboxypeptidase, not a beta-lactamase. A single amino acid change reveals evolutionary insights into beta-lactam resistance mechanisms in mycobacteria.

Area of Science:

  • Microbiology
  • Enzymology
  • Evolutionary Biology

Background:

  • Mycobacterium smegmatis possesses multiple penicillin-binding proteins (PBPs) and beta-lactamases, crucial for understanding beta-lactam resistance.
  • The enzyme MSMEG_6194 was annotated as a potential beta-lactamase, prompting investigation into its function and evolutionary significance.

Purpose of the Study:

  • To characterize the function of MSMEG_6194, a putative beta-lactamase from Mycobacterium smegmatis.
  • To investigate the evolutionary relationship between DD-carboxypeptidases and beta-lactamases in mycobacteria.

Main Methods:

  • Heterologous expression of MSMEG_6194 in E. coli and M. smegmatis deletion strains.
  • Biochemical assays including nitrocefin hydrolysis and pentapeptide substrate cleavage.
  • Structural analysis and site-directed mutagenesis (V139E mutant).

Main Results:

  • MSMEG_6194 exhibited no detectable beta-lactamase activity and did not confer beta-lactam resistance.
  • The enzyme demonstrated DD-carboxypeptidase activity, cleaving D-alanine from a pentapeptide substrate.
  • A V139E mutation restored beta-lactamase activity but reduced DD-carboxypeptidase function, highlighting altered catalytic preference.

Conclusions:

  • MSMEG_6194 primarily functions as a DD-carboxypeptidase.
  • A single amino acid substitution can shift enzymatic activity from DD-carboxypeptidase to beta-lactamase.
  • This study provides insights into the evolutionary pathways from DD-carboxypeptidases to beta-lactamases in mycobacteria, relevant to beta-lactam resistance.

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