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Amino acid variants at the P94 position in <i>Staphylococcus aureus</i> class A sortase modulate substrate binding and enzyme activity.

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Amino Acid Variants at the P94 Position in Staphylococcus aureus Class a Sortase Modulate Substrate Binding and

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Investigating mutations at the P94 position of Staphylococcus aureus sortase A (saSrtA) revealed variants with enhanced activity and altered substrate specificity for sortase-mediated ligation (SML). These findings suggest potential for improved SML applications through rational design.

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Area of Science:

  • Biochemistry
  • Microbiology
  • Enzymology

Background:

  • Gram-positive bacteria surface proteins are crucial for viability and pathogenicity.
  • Sortase enzymes anchor proteins to the peptidoglycan layer, making them potential antibiotic targets.
  • Sortase enzymes are utilized in sortase-mediated ligation (SML) for protein engineering.

Purpose of the Study:

  • To investigate the impact of single mutations at the P94 position of Staphylococcus aureus sortase A (saSrtA) on enzyme activity and substrate specificity.
  • To identify novel saSrtA variants with improved performance for SML applications.

Main Methods:

  • 18 P94X mutations (excluding cysteine) were created in saSrtA.
  • Enzyme activity was tested against four substrate sequences (LPATG, LPETG, LPKTG, LPSTG).
  • SaSrtA5M and P94D saSrtA5M variants were compared in a model SML reaction.

Main Results:

  • Several P94 variants exhibited higher activity and different substrate specificities compared to the P94R mutation.
  • P94A and P94D saSrtA5M variants showed >3-fold increased activity over saSrtA5M on certain substrates.
  • P94D saSrtA5M demonstrated approximately 2-fold greater product formation in a model SML reaction.

Conclusions:

  • Single mutations at the P94 position can significantly enhance saSrtA activity and alter substrate specificity.
  • Optimized sortase variants hold promise for advancing SML applications.
  • Combining rational design with high-throughput methods like directed evolution could yield superior sortase variants for SML.