Purification and characterization of aminoglycoside phosphotransferase APH(6)-Id, a streptomycin-inactivating enzyme
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...
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Veröffentlicht in: | Molecular and cellular biochemistry 2014-02, Vol.387 (1-2), p.207-216 |
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Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | 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
32
P-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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ISSN: | 0300-8177 1573-4919 |
DOI: | 10.1007/s11010-013-1886-1 |