BROAD-SPECTRUM AMINOGLYCOSIDE PHOSPHOTRANSFERASE TYPE-III FROM ENTEROCOCCUS - OVEREXPRESSION, PURIFICATION, AND SUBSTRATE-SPECIFICITY

The aminoglycoside phosphotransferases (APHs) are responsible for the bacterial inactivation of many clinically useful aminoglycoside antibi otics. We report the characterization of an enterococcal enzyme, APH(3 ')-IIIa, which inactivates a broad spectrum of aminoglycosides by ATP- dependent O-phosphorylation, Overproduction of APH(3')-IIIa has permit ted the isolation of 30-40 mg of pure protein/(L of cell culture). Pur ified APH(3')-IIIa is a mixture of monomer and dimer which is slowly c onverted to dimer only over time. Dimer could be dissociated into mono mer by incubation with 2-mercaptoethanol, suggesting that dimerization is mediated by formation of disulfide bond(s). Both monomer and dimer show K,values in the low micromolar range for good substrates such as kanamycin and neomycin, and k(cat) values of 1-4 s(-1). All aminoglyc osides show substrate inhibition except amikacin and kanamycin B. Dete rmination of minimum inhibitory concentrations indicates a positive co rrelation between antibiotic activity and k(cat)/K-m, but not with K-m or k(cat). NMR analysis of phosphorylated kanamycin A has directly de monstrated regiospecific phosphoryl transfer to the 3'-hydroxyl of the 6-aminohexose ring of the antibiotic. Analysis of structure-activity relationships with a variety of aminoglycosides has revealed that the deoxystreptamine aminocyclitol ring plays a critical role in substrate binding. This information will form the basis for future design of in hibitors of APH(3')-IIIa.