Substrate specificities and structure-activity relationships for acylation of antibiotics catalyzed by kanamycin acetyltransferase.

Antibiotic resistance caused by the presence of the plasmid pMH67 is mediated by the aminoglycoside acetyltransferase AAC(6')-4, also known as kanamycin acetyltransferase. Bacteria harboring the plasmid are resistant to the kanomycins plus a broad range of other deoxystreptamine-containing aminoglycosides but not to the gentamicins XK62-2 and C1 which are substituted at the 6'-position. Substrate specificity studies on the purified enzyme, however, now show that the enzyme acetylates an even broader range of aminoglycosides, including the gentamicins XK62-2 and C1. The enzyme also accepts several acyl-CoA esters, which differ in nucleotide as well as in acyl chain length. Application of the method of analysis of structure-activity data developed earlier for gentamicin acetyltransferase [Williams, J. W., & Northrop, D. B. (1978) J. Biol. Chem. 253, 5908-5914] to the kinetic data obtained for AAC(6')-4 shows that the turnover of the acylation reaction is limited by catalysis and not by the rate of release of either the acetylated antibiotic or CoA. Most structural changes in aminoglycosides cause changes in rates of release, and only drastic changes, near the 6'-amino group, affect catalysis. The structural requirements on aminoglycosides for enzymatic activity run parallel to the structural requirements for antibacterial activity.