STRUCTURE OF AN ANTIBIOTIC DEACTIVATING ENZYME. Lars C. Pedersen & Hazel M. Holden, University of Wisconsin, Madison, WI 53705

Kanamycin nucleotidyltransferase(KNTase) is a plasmid-coded enzyme which deactivates various antibiotics by transferring a nucleoside monophosphate group from ATP to the 4' hydroxyl group of the drug. Recently, we have solved the structure of KNTase in the presence of both kanamycin and AMPCPP. Crystals were grown from polyethylene glycol solutions and belonged to the space group P2₁2₁2₁ with unit cell dimensions of a=57.3 Å, b=102.2 Å, and c=101.8 Å and one dimer in the asymmetric unit. Least-squares refinement of the model at 2.5 Å resolution reduced the crystallographic R-factor to 16.8%. The binding pockets for both the nucleotide and the antibiotic are composed of residues from each subunit. There are few specific interactions between the protein and the adenine ring of the nucleotide. The majority of the nucleotide:protein interactions involve the phosphoryl oxygens and various side chain moieties. Kanamycin binds with the 4' hydroxyl group at 5 Å from the (-phosphorus of the nucleotide and is in the proper orientation for a single in-line displacement attack at the phosphorus. Based on the structure of the KNTase/AMPCPP/antibiotic complex, a possible mechanism for the enzyme has been proposed with Glu 145 serving as the active site base. This residue is in the proper position to extract the proton from the 4'-hydroxyl group thereby activating the kanamycin for subsequent attack at the (-phosphorus of the nucleotide. The Mg²⁺/pyrophosphate moiety would serve as an excellent leaving group. In addition, the close proximity of Lys 149 to one of the (-phosphoryl oxygens may increase the electrophilic character of the phosphorus center thus making it more susceptible to nucleophilic attack.

The overall goal of this investigation is to provide a structural framework by which new drugs may be designed. In addition, the elucidation of the mechanism of nucleotide phosphate transfer by KNTase could possibly serve as a model for the reactions catalyzed by DNA polymerases. Currently, we are in the process of testing our proposed mechanism by creating site-directed mutants of Glu 145 and Lys 149. Crystals of KNTase in the presence of both tobramycin and amikacin have also been obtained. Crystallographic analysis of KNTase in the presence of multiple antibiotics will help to address important protein:drug interactions.