Structural Recognition of Spectinomycin by Resistance Enzyme ANT(9) from Enterococcus faecalis

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Spectinomycin is a ribosome-binding antibiotic inhibiting protein translation.
• Aminoglycoside nucleotidyltransferases (ANTs) are key enzymes conferring resistance by modifying antibiotics like spectinomycin.
• ANT(9) specifically modifies spectinomycin, while ANT(3")(9) modifies both spectinomycin and streptomycin.

Purpose of the Study:

• To determine the structural mechanism of spectinomycin modification by ANT(9).
• To elucidate the structural basis for spectinomycin specificity among ANT enzymes.
• To understand the role of magnesium ions in the catalytic activity of ANT(9).

Main Methods:

• X-ray crystallography was used to solve the structures of ANT(9) in various states: apo, with ATP, with spectinomycin, and with both.
• Structural comparisons were made between ANT(9) and the related enzyme AadA (ANT(3")(9)).
• Analysis of active site residues and bound ligands, including magnesium ions.

Main Results:

• The first structures of spectinomycin bound to an ANT enzyme (ANT(9)) were determined.
• Spectinomycin binds near the interdomain cleft entrance, with ATP at the bottom; C-terminal domain rotation upon binding closes the cleft.
• Structural differences, including a straight α5 helix and shorter loop in ANT(9), explain spectinomycin specificity compared to streptomycin.
• Two magnesium ions were observed in the active site, one in a novel position potentially crucial for catalysis.
• The binding mode suggests spectinomycin analogues are also substrates for ANT(9) and ANT(3")(9).

Conclusions:

• The structures provide a detailed molecular understanding of how ANT(9) modifies spectinomycin.
• The findings explain the basis of antibiotic resistance mediated by ANT(9) and ANT(3")(9) enzymes.
• This structural insight may inform the design of novel antibiotics or strategies to overcome ANT-mediated resistance.