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Characterization of a key aminoglycoside phosphotransferase in gentamicin biosynthesis

Lei Shao1, Junsheng Chen, Chunxia Wang

  • 1State Key Lab of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, 1320 West Beijing Rd., Shanghai 200040, China.

Bioorganic & Medicinal Chemistry Letters
|January 24, 2013
PubMed

Insights

Gentamicin

Area of Science:

  • Microbiology
  • Biochemistry
  • Molecular Biology

Background:

  • Gentamicin, an aminoglycoside antibiotic, is crucial for treating bacterial infections.
  • Its 3'-hydroxyl group absence confers resistance to bacterial modification enzymes.
  • C-3' dehydroxylation is a key step in gentamicin biosynthesis.

Purpose of the Study:

  • To investigate the role of phosphorylation in the C-3' dehydroxylation step of gentamicin biosynthesis.
  • To clone, overexpress, and characterize the gentamicin biosynthetic gene gntI.
  • To confirm the enzymatic activity of the GntI protein.

Main Methods:

  • Cloning and overexpression of the gentamicin biosynthetic gene gntI in Escherichia coli.
  • Purification of the aminoglycoside phosphotransferase encoded by gntI.
  • Determination of kinetic parameters for various aminoglycoside substrates.
  • Structural elucidation using NMR spectroscopy of phosphorylated kanamycin B.

Main Results:

  • The gntI gene was successfully cloned and the GntI protein was purified.
  • NMR data confirmed regiospecific phosphorylation at the 3'-hydroxyl group of kanamycin B.
  • Kinetic parameters for Kanamycin A, Kanamycin B, Neomycin B, and Amikacin were determined.
  • These findings provide partial confirmation that 3'-dehydroxylation is preceded by 3'-phosphorylation.

Conclusions:

  • The GntI enzyme, an aminoglycoside phosphotransferase, plays a role in gentamicin biosynthesis.
  • 3'-phosphorylation is a likely prerequisite for the 3'-dehydroxylation step in gentamicin production.
  • GntI may represent a novel group of aminoglycoside phosphotransferases involved in antibiotic biosynthesis.

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