Site-specific mutations of conserved C-terminal residues in aminoglycoside 3'-phosphotransferase II: phenotypic and structural analysis of mutant enzymes.
In order to test the biological importance of amino acids in the C-terminal quarter of aminoglycoside 3'-phosphotransferase II enzyme, seven of the highly conserved residues in this region, His-188, Asp-190, Asp-208, Gly-210, Arg-211, Asp-216 and Asp-220, were changed via site-directed mutagenesis. The phenotype of each mutant was compared to wildtype in terms of antibiotic susceptibilities and enzymatic activities. All of the substitutions either abolished or significantly reduced the resistance of the resulting strains to kanamycin, neomycin, butirosin, ribostamycin, paromomycin, gentamicin A, and G-418. Similarly, enzyme activities in crude extracts were substantially reduced for the mutant strains. Affinity of the enzyme for Mg+2-ATP decreased with His-188, Asp-190, Asp-216 and Asp-220 substitutions as revealed by Km measurements. Secondary structure analysis predicted that substitutions at the conserved residues caused severe conformational distortions at the corresponding regions of the protein.
Kocabiyik
S
S
Department of Biology, University of Louisville, KY 40292.
Perlin
M H
MH
D000595:Amino Acid Sequence; D000617:Aminoglycosides / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D015153:Blotting, Western; D004251:DNA Transposable Elements; D019868:Kanamycin Kinase; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D000617:Aminoglycosides; D004251:DNA Transposable Elements; D010770:Phosphotransferases; D019868:Kanamycin Kinase