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Host-specific enzyme-substrate interactions in SPM-1 metallo-β-lactamase are modulated by second sphere residues

Lisandro J González1, Diego M Moreno2, Robert A Bonomo3

  • 1Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR) and Area Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.

Plos Pathogens
|January 7, 2014
PubMed

Insights

Mutations outside the active site of Pseudomonas aeruginosa metallo-β-lactamase (SPM-1) alter its resistance profile and zinc requirements. This molecular evolution enhances its effectiveness against beta-lactam antibiotics in clinical settings.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • Pseudomonas aeruginosa is a highly virulent and antibiotic-resistant Gram-negative pathogen.
  • Metallo-β-lactamases (MβLs) are enzymes conferring resistance to β-lactam antibiotics by hydrolysis.
  • SPM-1 is a unique MβL exclusively produced by P. aeruginosa, with atypical residues influencing its function.

Purpose of the Study:

  • To investigate how modifications of "atypical" second sphere residues (S84, G121) in SPM-1 affect its enzymatic activity and resistance profile.
  • To understand the role of these residues in substrate selectivity, zinc(II) dependency, and protein stability.
  • To explore how molecular evolution of SPM-1 can be optimized for the needs of P. aeruginosa during infection.

Main Methods:

  • Codon randomization was performed on the S84 and G121 positions of SPM-1.
  • Mutants were selected for enhanced resistance-conferring properties.
  • Minimum inhibitory concentrations (MICs), periplasmic enzymatic activity, Zn(II) requirements, and protein stability were assessed for wild-type and mutant SPM-1.

Main Results:

  • The identity of second sphere residues significantly modulates SPM-1's substrate preferences and resistance profile.
  • Wild-type SPM-1's second sphere residues confer substrate selectivity specific to the periplasmic environment.
  • Mutations allow SPM-1 to confer resistance even under Zn(II)-limiting conditions, relevant to infection environments.
  • Optimized catalytic efficiency, enzyme stability, and Zn(II) binding were achieved through substitutions outside the active site.

Conclusions:

  • Substitutions in the second sphere of SPM-1, outside the active site, are crucial for its function and adaptation.
  • Molecular evolution of SPM-1 can tailor its properties to meet the specific demands of the pathogenic host, including antibiotic resistance and nutrient availability.
  • These findings highlight a mechanism by which bacterial pathogens evolve to overcome therapeutic challenges.

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