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Purification and characterization of aminoglycoside phosphotransferase APH(6)-Id, a streptomycin-inactivating enzyme

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Abstract

As part of an overall project to characterize the streptomycin phosphotransferase enzyme APH(6)-Id, which confers bacterial resistance to streptomycin, we cloned, expressed, purified, and characterized the enzyme. When expressed in Escherichia coli, the recombinant enzyme increased by up to 70-fold the minimum inhibitory concentration needed to inhibit cell growth. Size-exclusion chromatography gave a molecular mass of 31.4 ± 1.3 kDa for the enzyme, showing that it functions as a monomer. Activity was assayed using three methods: (1) an HPLC-based method that measures the consumption of streptomycin over time; (2) a spectrophotometric method that utilizes a coupled assay; and (3) a radioenzymatic method that detects production of 32P-labeled streptomycin phosphate. Altogether, the three methods demonstrated that streptomycin was consumed in the APH(6)-Id-catalyzed reaction, ATP was hydrolyzed, and streptomycin phosphate was produced in a substrate-dependent manner, demonstrating that APH(6)-Id is a streptomycin phosphotransferase. Steady-state kinetic analysis gave the following results: K m(streptomycin) of 0.38 ± 0.13 mM, K m(ATP) of 1.03 ± 0.1 mM, V max of 3.2 ± 1.1 μmol/min/mg, and k cat of 1.7 ± 0.6 s−1. Our study demonstrates that APH(6)-Id is a bona fide streptomycin phosphotransferase, functions as a monomer, and confers resistance to streptomycin.

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Acknowledgments

We gratefully acknowledge support from the U. S. National Institutes of Health (NIH) RCMI program (Grant number 8G12MD007597) and MBRS-SCORE program (Grants number 3S06GM0816-33S1, SC3GM083752, and SC1GM08232). We especially thank Ms. Myrna Stupart of the Department of Microbiology at Howard University for help with the antibiotic susceptibility protocols and for providing E. coli control strain MM294.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, College of Medicine, Howard University, 520 W Street, NW, Washington, DC, 20059, USA

    Meseret Ashenafi & W. Malcolm Byrnes

  2. Center for Sickle Cell Disease and Department of Medicine, College of Medicine, Howard University, 520 W Street, NW, Washington, DC, 20059, USA

    Tatiana Ammosova & Sergei Nekhai

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  1. Meseret Ashenafi
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  2. Tatiana Ammosova
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  3. Sergei Nekhai
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  4. W. Malcolm Byrnes
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Correspondence to W. Malcolm Byrnes.

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Ashenafi, M., Ammosova, T., Nekhai, S. et al. Purification and characterization of aminoglycoside phosphotransferase APH(6)-Id, a streptomycin-inactivating enzyme. Mol Cell Biochem 387, 207–216 (2014). https://doi.org/10.1007/s11010-013-1886-1

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