Solution Studies of Isepamicin and Conformational Comparisons between Isepamicin and Butirosin A When Bound to an Aminoglycoside 6′-N-Acetyltransferase Determined by NMR Spectroscopy Enrico L. DiGiammarino, ‡ Kari-ann Draker, § Gerard D. Wright, § and Engin H. Serpersu* ,‡ Department of Biochemistry, Cellular and Molecular Biology, UniVersity of Tennessee, KnoxVille, Tennessee 37996-0840 and Department of Biochemistry, McMaster UniVersity, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5 ReceiVed NoVember 12, 1997; ReVised Manuscript ReceiVed January 12, 1998 ABSTRACT: NMR spectroscopy, combined with molecular modeling, was used to determine the conforma- tions of isepamicin and butirosin A in the active site of aminoglycoside 6′-N-acetyltranferase-Ii [AAC- (6′)-Ii]. The results suggest two enzyme-bound conformers for isepamicin and one for butirosin A. The dihedral angles that describe the glycosidic linkage between the A and B rings for the two conformers of AAC(6′)-Ii-bound isepamicin were φ AB )-7.9 ( 2.0° and ψ AB )-46.2 ( 0.6° for conformer 1 and φ AB )-69.4 ( 2.0° and ψ AB )-57.7 ( 0.5° for conformer 2. Unrestrained molecular dynamics calculations showed that these distinct conformers are capable of interconversion at 300 K. When superimposed at the 2-deoxystreptamine ring, one enzyme-bound conformer of isepamicin (conformer 1) places the reactive 6′ nitrogen in a similar position as that of butirosin A. Conformer 2 of AAC(6′)-Ii- bound isepamicin may represent an unproductive binding mode. Unproductive binding modes (to aminoglycoside modifying enzymes) could provide one reason isepamicin remains one of the more effective aminoglycoside antibiotics. The enzyme-bound conformation of butirosin A yielded an orthogonal arrangement of the 2,6-diamino-2,6-dideoxy-D-glucose and D-xylose rings, as opposed to the parallel arrangement which was observed for this aminoglycoside in the active site of an aminoglycoside 3′-O- phosphotransferase [Cox, J. R., and Serpersu, E. H. (1997) Biochemistry 36, 2353-2359]. The complete proton and carbon NMR assignments of the aminoglycoside antibiotic isepamicin at pH 6.8 as well as the pK a values for it’s amino groups are also reported. Aminoglycosides are a class of antibiotics used for treatment against staphylococci, a variety of Gram-negative bacteria and Gram-positive bacilli (1). Their primary target is the 16S rRNA of the 30S ribosomal subunit, binding to which results in disruption of normal protein biosynthesis, eventually leading to cell death. However, as with most antibiotics, bacterial resistance to aminoglycosides has become increasingly problematic (2). While there are several mechanisms by which resistance can arise, the most clinically relevant resistance mechanism is enzymatic inactivation of aminoglycosides by N-acetyltransferases (AAC), O-nucle- otidyltransferases (ANT), and O-phosphotransferases (APH) (3, 4). Over 50 different aminoglycoside modifying enzymes among the three classes have been identified (3). Many individual enzymes within each class can modify a broad range of aminoglycosides. Investigations of the modes by which aminoglycosides bind to their modifying enzymes may provide valuable insight into important structural and con- formational characteristics that give rise to broad substrate specificity. Two major families of aminoglycosides are disubstituted at positions 4 and 5 or 4 and 6 at the 2-deoxystreptamine ring. Isepamicin, a semisynthetic derivative of the gentami- cin B, and butirosin A were used in this study as representa- tives of the two major families of aminoglycosides (Figure 1). The A rings (or primed rings) in isepamicin and butirosin A are 6-amino-6-deoxy-D-glucose and 2,6-diamino-2,6- dideoxy-D-glucose, respectively. The B rings (or unprimed rings) are 2-deoxystreptamine modified at position N - 1 with an (S)-3-amino-2-hydroxypropionyl group in isepamicin and an (S)-4-amino-2-hydroxybutyryl group in butirosin A, which are denoted with the letter D (or triple-prime). The C ring (or double-primed ring) in isepamicin is D-garosamine and in butirosin A is D-xylose. The most common aminoglycoside modifying enzymes in pathogenic Gram-negative bacteria are the 6′-N-acetyl- transferases (5). Recently, a chromosomally encoded ami- noglycoside 6′-N-acetyltransferase [AAC(6′)-Ii] from En- terococcus faecium has been cloned, overexpressed, and characterized (6). This enzyme catalyzes the regiospecific acetyl transfer from acetylCoA to the 6 A (or 6′) nitrogen of aminoglycosides with free 6 A amine groups rendering these antibiotics inoffensive. The enzyme confers resistance to * To whom correspondence should be addressed. Phone: 423-974- 2668. Fax: 423-974-6306. E-mail: serpersu@bionmr.bio.utk.edu. ‡ University of Tennessee. § McMaster University. 1 Abbreviations: AAC, aminoglycoside acetyltransferase; ANT, aminoglycoside nucleotidyltransferase; APH, aminoglycoside phos- photransferase; COSY, correlated spectroscopy; HOHAH, Ahomo- nuclear Hartmann-Hahn spectroscopy; NOESY, nuclear Overhauser effect spectroscopy; rmsd, root-mean-square deviation; TRNOESY, transferred nuclear Overhauser effect spectroscopy. 3638 Biochemistry 1998, 37, 3638-3644 S0006-2960(97)02778-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 02/26/1998