Structural investigation of the antibiotic and ATP-binding sites in kanamycin nucleotidyltransferase

Abstract

Kanamycin nucleotidyltransferase (KNTase) is a plasmid-coded enzyme responsible for some types of bacterial resistance to aminoglycosides. The enzyme deactivates various antibiotics by transferring a nucleoside monophosphate group from ATP to the 4'-hydroxyl group of the drug. Detailed knowledge of the interactions between the protein and the substrates may lead to the design of aminoglycosides less susceptible to bacterial deactivation. Here we describe the structure of KNTase complexed with both the nonhydrolyzable nucleotide analog AMPCPP and kanamycin. Crystals employed in the investigation were grown from poly(ethylene glycol) solutions and belonged to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 57.3 A, b = 102.2 A, c = 101.8 A, and one dimer in the asymmetric unit. Least-squares refinement of the model at 2.5 A resolution reduced the crystallographic R factor to 16.8%. The binding pockets for both the nucleotide and the antibiotic are extensively exposed to the solvent and are composed of amino acid residues contributed by both subunits in the dimer. There are few specific interactions between the protein and the adenine ring of the nucleotide; rather the AMPCPP molecule is locked into position by extensive hydrogen bonding between the alpha-, beta-, and gamma-phosphates and protein side chains. This, in part, may explain the observation that the enzyme can utilize other nucleotides such as GTP and UTP. The 4'-hydroxyl group of the antibiotic is approximately 5 A from the alpha-phosphorus of the nucleotide and is in the proper orientation for a single in-line displacement attack at the phosphorus.(ABSTRACT TRUNCATED AT 250 WORDS)