Identification, functional and structural characterization of novel aminoglycoside phosphotransferase APH(3″)-Id from Streptomyces rimosus subsp. rimosus ATCC 10970

Arch Biochem Biophys. 2019 Aug 15;671:111-122. doi: 10.1016/j.abb.2019.06.008. Epub 2019 Jun 26.

Abstract

In this study, we identified a new gene (aph(3″)-Id) coding for a streptomycin phosphotransferase by using phylogenetic comparative analysis of the genome of the oxytetracycline-producing strain Streptomyces rimosus ATCC 10970. Cloning the aph(3″)-Id gene in E.coli and inducing its expression led to an increase in the minimum inhibitory concentration of the recombinant E.coli strain to streptomycin reaching 350 μg/ml. To evaluate the phosphotransferase activity of the recombinant protein APH(3″)-Id we carried out thin-layer chromatography of the putative 32P-labeled streptomycin phosphate. We also performed a spectrophotometric analysis to determine the production of ADP coupled to NADH oxidation. Here are the kinetic parameters of the streptomycin phosphotransferase APH(3″)-Id: Km 80.4 μM, Vmax 6.45 μmol/min/mg and kcat 1.73 s-1. We demonstrated for the first time the ability of the aminoglycoside phototransferase (APH(3″)-Id) to undergo autophosphorylation in vitro. The 3D structures of APH(3″)-Id in its unliganded state and in ternary complex with streptomycin and ADP were obtained. The structure of the ternary complex is the first example of this class of enzymes with bound streptomycin. Comparison of the obtained structures with those of other aminoglycoside phosphotransferases revealed peculiar structure of the substrate-binding pocket reflecting its specificity to a particular antibiotic.

Keywords: 3D structure; APH(3″)-Id; Aminoglycoside phosphotransferase; Antibiotic resistance; Streptomyces rimosus; Streptomycin phosphotransferase; X-ray.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Anti-Bacterial Agents / pharmacology
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Computational Biology
  • Escherichia coli / drug effects
  • Escherichia coli / genetics
  • Genes, Bacterial
  • Phosphorylation
  • Phosphotransferases (Alcohol Group Acceptor) / chemistry*
  • Phosphotransferases (Alcohol Group Acceptor) / genetics
  • Phosphotransferases (Alcohol Group Acceptor) / isolation & purification
  • Phylogeny
  • Protein Processing, Post-Translational
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Sequence Alignment
  • Streptomyces rimosus / enzymology*
  • Streptomycin / pharmacology

Substances

  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Recombinant Proteins
  • Phosphotransferases (Alcohol Group Acceptor)
  • streptomycin 3''-kinase
  • Streptomycin