Tetracycline-Inactivating Enzymes

Abstract

Tetracyclines have been foundational antibacterial agents for more than 70 years. Renewed interest in tetracycline antibiotics is being driven by advancements in tetracycline synthesis and strategic scaffold modifications designed to overcome established clinical resistance mechanisms including efflux and ribosome protection. Emerging new resistance mechanisms, including enzymatic antibiotic inactivation, threaten recent progress on bringing these next-generation tetracyclines to the clinic. Here we review the current state of knowledge on the structure, mechanism, and inhibition of tetracycline-inactivating enzymes.

Keywords: antibiotic adjuvants; antibiotic resistance; enzymatic antibiotic inactivation; flavin monooxygenase; tetracycline destructases; tetracyclines.

FIGURE 1

Evolution of the tetracycline scaffold. 6-Deoxy-6-demethyltetracycline represents the minimum tetracycline pharmacophore required for inhibition of the ribosome. Tetracycline (first reported in 1953), CTc (first reported in 1948), and oxytetracycline (first reported in 1950) represent first generation structures. Metacycline (first reported in 1962), doxycycline (first reported in 1967), and minocycline (first reported in 1961) represent second generation structures. Tigecycline (first reported in 1993) is the only FDA-approved third generation structure, while omadacycline (first reported in 2013) and eravacycline (first reported in 2013) are fourth generation molecules currently in phase III clinical trials.

FIGURE 2

Molecular mechanisms of tetracycline resistance. (A) Efflux, exclusion, (B) ribosome protection, (C) ribosome modification, and (D) enzymatic inactivation. Documented ARGs associated with each type of tetracycline resistance are provided.

FIGURE 3

(A) Hydroxylation of oxytetracycline by TetX. (B) Mechanism of class A FMOs.

FIGURE 4

X-ray crystal structure of a tetracycline destructase with bound tetracycline substrate and flavin cofactor. The mobility of the flavin cofactor is highlighted by showing the FAD-IN and FAD-OUT conformations observed during structural studies. (A) X-ray crystal structure of CTc bound to TetX (FAD-IN conformation, PDB ID: 2y6r). (B) X-ray crystal structure of Tet50 with no bound substrate (FAD-OUT conformation, PDB ID: 5tue). (C) X-ray crystal structure of Tet50 with no bound substrate (FAD-IN conformation, PDB ID: 5tue). (D) X-ray crystal structure of CTc bound to Tet50 (FAD-IN conformation, PDB ID: 5tui). (E) Surface view of X-ray crystal structure of CTc bound to TetX (FAD-IN conformation, PDB ID: 2y6r). (F) Surface view of X-ray crystal structure of Tet50 with no bound substrate (FAD-OUT conformation, PDB ID: 5tue). (G) Surface view of X-ray crystal structure of Tet50 with no bound substrate (FAD-IN conformation, PDB ID: 5tue). (H) Surface view of X-ray crystal structure of CTc bound to Tet50 (FAD-IN conformation, PDB ID: 5tui). Images were generated using PyMOL v1.7.

FIGURE 5

(A) X-ray crystal structure of CTc bound to TetX in binding mode I_D,A defines the orientation of FAD relative to each CTc binding mode (PDB ID: 2y6r). (B) Theoretical and experimentally observed tetracycline binding modes (four total). Image in panel (A) was generated using PyMOL v1.7.

FIGURE 6

Recognition elements of CTc A-ring for each experimentally observed substrate-binding mode. (A) X-ray structure of CTc bound to TetX in Mode I_D,A (PDB ID: 2y6r). (B) Expanded X-ray structure of CTc bound to TetX in Mode I_D,A with interacting structural residues highlighted and labeled (PDB ID: 2y6r). (C) X-ray structure of CTc bound to Tet50 in Mode II_A,D (PDB ID: 5tui). (D) Expanded X-ray structure of CTc bound to Tet50 in Mode II_A,D with interacting structural residues highlighted and labeled (PDB ID: 5tui). Images were generated using PyMOL v1.7.

FIGURE 7

Victim of fate: the site of tetracycline oxidation is determined by binding mode and distance from flavin-C4a. Bond distances to reactive centers on CTc bound to TetX in Mode I_D,A (PDB ID: 2y6r) and CTc bound to Tet50 in Mode II_A,D (PDB ID: 5tui) were determined in PyMOL from the corresponding PDB files. Images of FAD were generated using PyMOL v1.7.

FIGURE 8

Cascade reactions leading to tetracycline degradation products from enzymatic C12-oxidation of mode I_D,A-bound tetracycline.

FIGURE 9

Cascade reactions leading to tetracycline degradation products from enzymatic C1- or C3-oxidation of mode II_A,D-bound tetracycline.

FIGURE 10

Alternative mechanistic pathway leading to formation of the ring contracted degradation product ([M+H]⁺ 467.1216) initiated by hydroxylation of C2.

FIGURE 11

(A) Structures of tetracycline (top) and anhydrotetracycline (bottom). Conformation of tetracycline (B) and anhydrotetracycline (C) as viewed from face and edge of the tetracyclic core. 3D structures of tetracycline and anhydrotetracycline in panels (B) and (C) were energy minimized using Spartan and images were generated using Mercury software v3.10.

FIGURE 12

(A) X-ray crystal structure of anhydrotetracycline bound to Tet50 in Mode I_A,D (PDB accession number 5tuf). (B) Surface view of X-ray crystal structure of aTC bound to Tet50. (C) X-ray crystal structure of anhydrotetracycline bound to Tet50 in Mode I_A,D with recognition residues highlighted. (D) Expanded X-ray crystal structure of anhydrotetracycline bound to Tet50 in Mode I_A,D with recognition residues highlighted and labeled. Images were generated using PyMOL v1.7.

FIGURE 13

A mechanistic model for the tetracycline destructase catalytic cycle and inhibition by anhydrotetracycline is proposed. (I) Flavin oxidized, open active site; (II) substrate binding, flavin oxidized, open active site; (III) substrate bound, flavin reduced, open active site; (IV) substrate bound with C4a-peroxyflavin in “in” conformation, closed active site; (V) oxidized product bound with C4a-hydroxyflavin in “in” conformation, closed active site; (VI) substrate bound with C4a-hydroxyflavin in “out” conformation, open active site; (VII) inhibitor bound with flavin in “out” conformation, open active site; (VIII) inhibitor bound with C4a-peroxyflavin in “in” conformation, closed active site.

FIGURE 14

(A) X-ray structure of tigecycline bound to TetX in Mode I_D,A (PDB accession number 4a6n). (B) Surface view of X-ray structure of tigecycline bound to TetX in Mode I_D,A. (C) X-ray structure of tigecycline bound to TetX in Mode I_D,A with relevant substrate recognition interactions highlighted. (D) Expanded X-ray structure of tigecycline bound to TetX in Mode I_D,A with relevant substrate recognition interactions highlighted for the A-ring (Q192, R213) and the D-ring N-t-butyl-glycylamide substituent (E367). Electron density for the C2-carboxamide bond was missing in the PDB file 4a6n. The C2-carboxamide bond was added using the create bond function in PyMOL. Images were generated using PyMOL v1.7.