Modification of APH(3')-IIIa, an aminoglycoside antibiotic 3'-phosphotranferase, to characterize its active site residues

Bacterial resistance to antibiotic drugs is posing a medical crisis situation to public health. Infections caused by the gram-positive genera represent the most important ones due to the fact that they are responsible for a wide variety of hospital-acquired infections. Treatment of staphylococcal and enterococcal infections most often require the administration of aminoglycoside/aminocyclitol group of antibiotics, which target the bacterial 308 ribosomal subunit, thereby interfering with the normal protein translation processes and ultimately leading to cell death. However, bacteria have responded to the pressure brought about by the use of these antibiotics by developing a number of mechanisms. One of the most common ways in which bacteria evade the toxic effects of these drugs is expression of an array of enzymes that chemically modify the antibiotic targets and thus render them inoffensive to the These detoxifying enzymes have been classified in three groups: aminoglycoside 0-phosphotransferases (APH family), 0-nucleotidyltransferases (ANT family), and the N-acetyltransferases (AAC family) of which the APH family of proteins are by far more commonly distributed in the pathogenic bacteria.

APH(3')-ΙΙΙa belongs to the APH group of enzymes with the broadest substrate specificity among all known seven isozymes. It has been recently overexpressed and characterized to be an aminoglycoside kinase, which regiospecifically transfers a phosphate group to the aminoglycoside substrate in an ATP-dependent fashion. Various kinetic studies have been performed on this enzyme in order to determine its catalytic mechanism.

Chemical modification of APH (3')-ΙΙΙa using DEPC to label His residues suggests that His residues are not involved in the catalytic activity. On the other hand, chemical modification of Tyr residues using TNM results in a specific modification of a single Tyr residue that causes a concurrent loss of enzymatic activity. Among the two substrates, only the aminoglycoside substrate protects the enzyme against inactivation by TNM at low substrate concentrations, whereas both Mg⁺²-ATP and the aminoglycoside substrate protect the enzyme at high substrate concentrations.

In order to determine the active site amino acid residues, the TNM-modified enzyme was tryptically digested and purified over reverse-phase HPLC column. The purified peptide was analyzed for its amino acid content using one-dimensional ¹H NMR method. The NMR analysis suggests that the purified active site peptide is 8 amino acid long with a single modified Tyr residue, determined from the sequence of the enzyme to be Tyr⁵⁵.

This work is significant in that it demonstrates, for the first time, the amino acid residues that are located at the active site of the enzyme and are involved in its catalytic activity. Furthermore, it brings insights into our understanding of the possible substrate-enzyme interactions for the ultimate goal of design of inhibitors of APH (3')-ΙΙΙa.