Aminoglycoside 2''-phosphotransferases mediate high level resistance to aminoglycoside antibiotics in Gram-positive microorganisms, thus posing a serious threat to the treatment of serious enterococcal infections. This work reports on cloning, purification, and detailed mechanistic characterization of aminoglycoside 2''-phosphotransferase, known as type Ic enzyme. In an unexpected finding, the enzyme exhibits strong preference for guanosine triphosphate over adenosine triphosphate as the phosphate donor, a unique observation among all characterized aminoglycoside phosphotransferases. The enzyme phosphorylates only certain 4,6-disubstituted aminoglycosides exclusively at the 2''-hydroxyl with k(cat) values of 0.5-1.0 s(-1) and K(m) values in the nanomolar range for all substrates but kanamycin A. Based on this unique substrate profile, the enzyme is renamed aminoglycoside 2''-phosphotransferase type IIIa. Product and dead-end inhibition patterns indicated a random sequential Bi Bi mechanism. Both the solvent viscosity effect and determination of the rate constant for dissociation of guanosine triphosphate indicated that at pH 7.5 the release of guanosine triphosphate is rate-limiting. A computational model for the enzyme is presented that sheds light on the structural aspects of interest in this family of enzymes.