Publications

• Enzymatic Adenylylation of Streptomycin and Spectinomycin by R-Factor-Resistant Escherichia coli.

  Benveniste R., Yamada T., Davies J.

  A resistance (R) factor-containing strain of Escherichia coli which is known to inactivate streptomycin by adenylylation has been shown to be spectinomycin resistant. An osmotic shockate of this strain catalyzes the formation of the biologically inactive spectinomycin adenylate, in which the adeny ... >> More

  A resistance (R) factor-containing strain of Escherichia coli which is known to inactivate streptomycin by adenylylation has been shown to be spectinomycin resistant. An osmotic shockate of this strain catalyzes the formation of the biologically inactive spectinomycin adenylate, in which the adenylyl residue is probably attached to a d-threo methylamino alcohol moiety of spectinomycin. Both the streptomycin and spectinomycin adenylylating activities show the same temperature inactivation profile, and both are present in a protein fraction purified for the streptomycin inactivating enzyme. Mutants obtained from this strain which were sensitive to either spectinomycin or streptomycin were shown to lack both enzymatic activities when tested in vitro. Revertants of these mutants selected for recovery of either streptomycin resistance or spectinomycin resistance regain both activities. Therefore, we conclude that the inactivation of the two drugs is catalyzed by the same enzyme. Examination of a number of R factor-carrying strains has shown that those strains which are resistant to streptomycin and spectinomycin contain the adenylylating enzyme, whereas strains resistant to streptomycin but sensitive to spectinomycin inactivate streptomycin by phosphorylation. << Less

  Infect Immun 1:109-119(1970) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural mechanism of AadA, a dual-specificity aminoglycoside adenylyltransferase from Salmonella enterica.

  Stern A.L., Van der Verren S.E., Kanchugal P S., Nasvall J., Gutierrez de Teran H., Selmer M.

  Streptomycin and spectinomycin are antibiotics that bind to the bacterial ribosome and perturb protein synthesis. The clinically most prevalent bacterial resistance mechanism is their chemical modification by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). A ... >> More

  Streptomycin and spectinomycin are antibiotics that bind to the bacterial ribosome and perturb protein synthesis. The clinically most prevalent bacterial resistance mechanism is their chemical modification by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). AadA from <i>Salmonella enterica</i> is an aminoglycoside (3″)(9) adenylyltransferase that <i>O-</i>adenylates position 3″ of streptomycin and position 9 of spectinomycin. We previously reported the apo-AadA structure with a closed active site. To clarify how AadA binds ATP and its two chemically distinct drug substrates, we here report crystal structures of WT AadA complexed with ATP, magnesium, and streptomycin and of an active-site mutant, E87Q, complexed with ATP and streptomycin or the closely related dihydrostreptomycin. These structures revealed that ATP binding induces a conformational change that positions the two domains for drug binding at the interdomain cleft and disclosed the interactions between both domains and the three rings of streptomycin. Spectinomycin docking followed by molecular dynamics simulations suggested that, despite the limited structural similarities with streptomycin, spectinomycin makes similar interactions around the modification site and, in agreement with mutational data, forms critical interactions with fewer residues. Using structure-guided sequence analyses of ANT(3″)(9) enzymes acting on both substrates and ANT(9) enzymes active only on spectinomycin, we identified sequence determinants for activity on each substrate. We experimentally confirmed that Trp-173 and Asp-178 are essential only for streptomycin resistance. Activity assays indicated that Glu-87 is the catalytic base in AadA and that the nonadenylating E87Q mutant can hydrolyze ATP in the presence of streptomycin. << Less

  J. Biol. Chem. 293:11481-11490(2018) [PubMed] [EuropePMC]

• Cloning and nucleotide base sequence analysis of a spectinomycin adenyltransferase AAD(9) determinant from Enterococcus faecalis.

  Leblanc D.J., Lee L.N., Inamine J.M.

  Enterococcus faecalis LDR55, a human clinical isolate, is resistant to tetracycline (Tcr), erythromycin (Emr), and high levels (greater than 2,000 micrograms/ml) of spectinomycin (Spr) but not streptomycin. Filter matings between strain LDR55 and E. faecalis OG1-RF produced transconjugants with th ... >> More

  Enterococcus faecalis LDR55, a human clinical isolate, is resistant to tetracycline (Tcr), erythromycin (Emr), and high levels (greater than 2,000 micrograms/ml) of spectinomycin (Spr) but not streptomycin. Filter matings between strain LDR55 and E. faecalis OG1-RF produced transconjugants with the following resistance phenotypes: Tcr Emr Spr, Tcr Emr, Tcr Spr, and Tcr only but never Emr or Spr only. The genetic determinant encoding resistance to spectinomycin was cloned in Streptococcus sanguis Challis from pDL55, a 26-kb plasmid harbored by a Tcr Spr transconjugant. Subcloning experiments yielded a 1.1-kb ClaI-NdeI fragment that encoded very high-level Spr in S. sanguis (10 mg/ml) and Escherichia coli (50 mg/ml). Cell extracts of cultures obtained from Spr strains expressed adenylating activity for spectinomycin but not for streptomycin, indicating that Spr was due to an AAD(9) activity. The nucleotide base sequence of the 1.1-kb ClaI-NdeI fragment contained a single 750-base open reading frame. The protein predicted from the open reading frame consisted of 250 amino acids and had a calculated size of approximately 28,000 daltons, similar to the size estimated from maxicell analysis (29,000 daltons). The deduced amino acid sequence of the streptococcal AAD(9) was compared with that of the AAD(9) encoded by staphylococcal transposon Tn554. The two proteins shared approximately 39% amino acid identity, which was expanded to 53% when conservative amino acid changes were included. When the streptococcal protein was compared with an AAD(3")(9) protein of E. coli, the degrees of identity were 27 and 47%, on the basis of actual amino acids and conservative replacements, respectively. The cloning and nucleotide base sequence analyses of the spectinomycin AAD(9) determinant from E. faecalis that results in high-level Spr when transferred to S. sanguis or E. coli are presented. << Less

  Antimicrob. Agents Chemother. 35:1804-1810(1991) [PubMed] [EuropePMC]

• Plasmid-determined resistance to antimicrobial agents.

  Davies J., Smith D.I.

  Annu Rev Microbiol 32:469-518(1978) [PubMed] [EuropePMC]