Regiospecificity assignment for the reaction of kanamycin nucleotidyltransferase from Staphylococcus aureus.
Aminoglycoside nucleotidyltransferases catalyze the transfer of a nucleoside monophosphoryl group from a nucleotide to a hydroxyl group of an aminoglycoside antibiotic. Kanamycin nucleotidyltransferase [ANT (4',4' ')-I] from Staphylococcus aureus confers resistance to numerous aminoglycosides with a 4' or 4' ' hydroxyl group in the equatorial position. The synthesis of m-nitrobenzyl triphosphate, a new substrate of kanamycin nucleotidyltransferase, is reported. The kanamycin nucleotidyltransferase catalyzed reaction of kanamycin A with m-nitrobenzyl triphosphate is 2 orders of magnitude slower than that with ATP. The MALDI-TOF spectra of the purified products of both reactions revealed that kanamycin A was modified only at one position. The regiospecificity of the reaction catalyzed by kanamycin nucleotidyltransferase of kanamycin A with either ATP or m-nitrobenzyl triphosphate was determined directly by one- and two-dimensional hetero- and homonuclear NMR techniques. The site of the modification was unambiguously assigned to the 4' hydroxyl of kanamycin A; thus, the products formed are 4'-(adenosine-5'-phosphoryl)-kanamycin A and 4'-(m-nitrobenzyl phosphoryl)-kanamycin A. This eliminates the uncertainty concerning the point of modification since this could not be determined from the crystal structure of the enzyme with bound MgAMPCPP and kanamycin A [Pedersen, L. C., Benninig, M. M., and Holden, H. M. (1995) Biochemistry 34, 13305-13311].
Institute for Enzyme Research, Department of Biochemistry, College of Agricultural Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.
B
B
Gerratana
W W
WW
Cleland
L A
LA
Reinhardt
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000302:isolation & purification; D007612:Kanamycin / Q000031:analogs & derivatives / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity
C425063:4'-(adenosine-5'-phosphoryl)-kanamycin A; C425064:4'-(m-nitrobenzyl phosphoryl)-kanamycin A; C425065:m-nitrobenzyl triphosphate; D000249:Adenosine Monophosphate; D007612:Kanamycin; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; C024251:kanamycin nucleotidyltransferase