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Photoaffinity labeling of Aminoglycoside Phosphotransferase 3′‐II confirms the involvement of conserved lysine in ATP binding
Author(s) -
Dholakia Jaydev N,
Brown Scott A,
Perlin Michael H
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a631
Subject(s) - biochemistry , phosphotransferase , binding site , enzyme , aminoglycoside , conserved sequence , chemistry , amino acid , active site , lysine , biology , peptide sequence , gene , antibiotics
Of the mechanisms bacteria use to evade the effects of aminoglycoside antibiotics, enzymatic modification has the most clinical relevance due to the promiscuous nature of the DNA encoding the genes for these enzymes. One such enzyme, aminoglycoside 3′‐phosphotransferase IIa (APH(3′)‐IIa), is used as a model for understanding this modification at a molecular level along with anticipating the evolution of AG resistance. We previously determined the crystal structure of APH(3′)‐IIa and proposed its ATP binding domain ( J Mol Biol 327 , , 2003). To better understand the function of the specific amino acids involved in substrate binding, we have generated a site‐specific mutant (K50R) and analyzed the kinetics for ATP binding. We also employed active‐site labeling using azido ATP analogs to isolate and sequence a peptide involved in ATP binding. Competition experiments during active‐site labeling revealed that a conserved tryptophan residue in the amino‐terminus of the enzyme may also be involved directly or by association with ATP binding. Together, these data give us a clearer picture of how ATP associates with APH(3′)‐II and could aid in the development of small chemical compounds which specifically inhibit its activity.