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

Abstract

The product of a kanamycin resistance gene encoded by plasmid pTB913 isolated from a thermophilic bacillus [Bacillus subtilis or B. stearothermophilus] 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, .apprx. 1200 base pairs were sequenced, followed by amino-terminal amino acid sequencing of the enzyme. The 2 nucleotide sequences were identical to each other except for only 1 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 (MW, .apprx. 29,000). The sole difference was transversion from a cytosine (pUB110) to an adenine (pTB913) at a position +389, counting the 1st 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 2 enzymes caused by a single amino acid replacement is discussed in light of electrostatic effects.