Enzymatic and nucleotide sequence studies of a kanamycin-inactivating enzyme encoded by a plasmid from thermophilic bacilli in comparison with that encoded by plasmid pUB110.
The product of a kanamycin resistance gene encoded by plasmid pTB913 isolated from a thermophilic bacillus was identified as a kanamycin nucleotidyltransferase which is similar to that encoded by plasmid pUB110 from a mesophile, Staphylococcus aureus. The enzyme encoded by pTB913 was more thermostable than that encoded by pUB110. In view of a close resemblance of restriction endonuclease cleavage maps around the BglII site in the structural genes of both enzymes, ca. 1,200 base pairs were sequenced, followed by amino-terminal amino acid sequencing of the enzyme. The two nucleotide sequences were found to be identical to each other except for only one base in the midst of the structural gene. Each structural gene, initiating from a GUG codon as methionine, was composed of 759 base pairs and 253 amino acid residues (molecular weight, ca. 29,000). The sole difference was transversion from a cytosine (pUB110) to an adenine (pTB913) at a position + 389, counting the first base of the initiation codon as + 1. That is, a threonine at position 130 for the pUB110-coded kanamycin nucleotidyltransferase was replaced by a lysine for the pTB913-coded enzyme. The difference in thermostability between the two enzymes caused by a single amino acid replacement is discussed in light of electrostatic effects.
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics*; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004352:Drug Resistance, Microbial; D005796:Genes; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics*; D007612:Kanamycin / Q000633:toxicity; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D011815:R Factors; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics*
D007612:Kanamycin; D009713:Nucleotidyltransferases; C024251:kanamycin nucleotidyltransferase; D004262:DNA Restriction Enzymes