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Structural recognition of spectinomycin by resistance enzyme ANT(9) from Enterococcus faecalis

DOI: 10.1128/AAC.00371-20 DOI Help

Authors: Sandesh Kanchugal Puttaswamy (Uppsala University) , Maria Selmer (Uppsala University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Antimicrobial Agents And Chemotherapy

State: Published (Approved)
Published: April 2020
Diamond Proposal Number(s): 15868 , 22906

Abstract: Spectinomycin is a ribosome-binding antibiotic that blocks the translocation step of translation. A prevalent resistance mechanism is the modification of the drug by aminoglycoside nucleotidyl transferase (ANT) enzymes of the spectinomycin-specific ANT (9) family or by the dual-specificity ANT(3") (9) family that also acts on streptomycin. We previously reported the structural mechanism of streptomycin modification by the ANT(3") (9) AadA from Salmonella enterica. ANT (9) from Enterococcus faecalis adenylates the 9-hydroxyl of spectinomycin. We here present the first structures of spectinomycin bound to an ANT enzyme. Structures were solved for ANT (9) in apo form, in complex with ATP, spectinomycin and magnesium or in complex with only spectinomycin. ANT (9) shows similar overall structure as AadA with an N-terminal nucleotidyltransferase domain and a C-terminal α-helical domain. Spectinomycin binds close to the entrance of the interdomain cleft, while ATP is buried at the bottom. Upon drug binding, the C-terminal domain rotates by 14 degrees to close the cleft, allowing contacts of both domains with the drug. Comparison with AadA shows that spectinomycin specificity is explained by a straight α5 helix and a shorter α5-α6 loop that would clash with the larger streptomycin substrate. In the active site, we observe two magnesium ions, one of them in a previously un-observed position that may activate the 9-hydroxyl for deprotonation by the catalytic base Glu-86. The observed binding mode for spectinomycin suggests that also spectinamides and aminomethyl spectinomycins, recent spectinomycin analogues with expansions in position 4 of the C ring, will be subjected to modification by ANT (9) and ANT(3") (9) enzymes.

Journal Keywords: antibiotic resistance; spectinomycin; enzyme structure; X-ray crystallography; antimicrobial agents

Subject Areas: Biology and Bio-materials, Medicine


Instruments: I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography