Structural and functional characterization of C39 endoisopeptidases from methanogenic archaeal phages with activity

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Leizhou Guo1, Huan Leng2, Xin Zhou1

  • 1Key Laboratory of Development and Application of Rural Renewable Energy, Biogas Institute of Ministry of Agriculture and Rural Affairs, Chengdu, Sichuan, 610041, China.

International Journal of Biological Macromolecules
|May 22, 2026
PubMed

Insights

Researchers investigated archaeal cell wall hydrolases, specifically C39 peptidases from methanogenic archaeal phages. Structural analysis revealed key differences from bacterial enzymes, identifying novel pseudopeptidoglycan endoisopeptidases.

Area of Science:

  • Microbiology
  • Structural Biology
  • Biochemistry

Background:

  • Archaeal pseudopeptidoglycan offers inherent resistance to common antibiotics and lysozyme.
  • Methanogenic archaeal phages encode C39 peptidases with potential cell wall hydrolysis activity.
  • Bacterial C39 peptidases differ functionally, processing double glycine motifs in ABC transport systems.

Purpose of the Study:

  • To characterize the substrate specificities and catalytic mechanisms of phage-derived C39 peptidases.
  • To elucidate the structural basis for functional differences between archaeal and bacterial C39 peptidases.
  • To classify C39 peptidases and identify novel enzymes targeting archaeal cell walls.

Main Methods:

  • Enzymatic assays using synthetic isopeptide and p-nitroaniline-modified peptide substrates.
  • X-ray crystallography to determine enzyme structures.
  • Site-directed mutagenesis to probe functional determinants.

Main Results:

  • Three phage-derived C39 peptidases (C39-PeiR, C39-PhiF1, C39-PhiF3) were characterized.
  • A specific loop region adjacent to α-helix 7 in C39-PeiR was identified as a key structural determinant for function.
  • Structural variations led to the classification of C39 peptidases into 16 clades, revealing novel pseudopeptidoglycan endoisopeptidases.

Conclusions:

  • Phage-derived C39 peptidases possess distinct substrate specificities and catalytic properties compared to bacterial orthologs.
  • Structural insights highlight the role of specific loop regions in enzyme function and host specificity.
  • This study expands the known repertoire of enzymes targeting archaeal cell walls, with implications for antimicrobial development.
Keywords:
C39 peptidaseCatalytic mechanismMethanogenic archaeaPseudopeptidoglycan

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