Multiple Keys for a Single Lock: The Unusual Structural Plasticity of the Nucleotidyltransferase (4′)/Kanamycin Complex

Abstract

The most common mode of bacterial resistance to aminoglycoside antibiotics is the enzyme-catalysed chemical modification of the drug. Over the last two decades, significant efforts in medicinal chemistry have been focused on the design of non- inactivable antibiotics. Unfortunately, this strategy has met with limited success on account of the remarkably wide substrate specificity of aminoglycoside-modifying enzymes. To understand the mechanisms behind substrate promiscuity, we have performed a comprehensive experimental and theoretical analysis of the molecular-recognition processes that lead to antibiotic inactivation by Staphylococcus aureus nucleotidyltransferase 4'(ANT(4')), a clinically relevant protein. According to our results, the ability of this enzyme to inactivate structurally diverse polycationic molecules relies on three specific features of the catalytic region. First, the dominant role of electrostatics in aminoglycoside recognition, in combination with the significant extension of the enzyme anionic regions, confers to the protein/antibiotic complex a highly dynamic character. The motion deduced for the bound antibiotic seem to be essential for the enzyme action and probably provide a mechanism to explore alternative drug inactivation modes. Second, the nucleotide recognition is exclusively mediated by the inorganic fragment. In fact, even inorganic triphosphate can be employed as a substrate. Third, ANT(4') seems to be equipped with a duplicated basic catalyst that is able to promote drug inactivation through different reactive geometries. This particular combination of features explains the enzyme versatility and renders the design of non-inactivable derivatives a challenging task.

Comparing aminoglycoside binding sites in bacterial ribosomal RNA and aminoglycoside modifying enzymes

“…If the drug was forced to bind to the binary complex ANT:ATP, then it would probably encounter difficulties in adapting its conformation to the nonflexible binding site. The recent study of the binary and ternary complexes of ANT(4′)86 has provided a new hypothesis for the mechanism of binding of the substrates. According to that study, the aminoglycoside first binds to a nonspecific binding site, and the drug only relocates to the final binding cleft when a nucleotide is present.…”

Revealing the Structural Basis of Promiscuous Amine Transaminase Activity

“…In contrast to enzymes that provide multiple binding sites for different substrates,1, 2 TAs have only one site for binding different amino acids, that is, they have developed dexterous mechanisms to adapt the active site to fit chemically different substrates 14. This ability—that one single “lock” accepts multiple “keys”15—is known as dual substrate recognition (Scheme ).…”

Structure of AadA fromSalmonella enterica: a monomeric aminoglycoside (3′′)(9) adenyltransferase

“…These results indicate that binding of ATP and magnesium between the two domains of AadA will orient the two domains for binding of either of the aminoglycoside substrates to the intersubunit pocket and that the O atom between theand -phosphates forms a critical interaction with AadA. Thus, AadA binds ATP before the aminoglycoside substrate, in contrast to KNTase, where kanamycin first binds to a lower affinity nonspecific binding site and then relocates to the final binding cleft when a nucleotide is present (Matesanz et al, 2012). This also agrees with the pre-formed ATP-binding site in the dimeric apo KNTase structure and the closed ATP-binding site of the present apo AadA structure.…”