The Tetracycline Destructases: A Novel Family of Tetracycline-Inactivating Enzymes

Abstract

Summary Enzymes capable of inactivating tetracycline are paradoxically rare compared with enzymes that inactivate other natural product antibiotics. We describe a family of flavoenzymes, previously unrecognizable as resistance genes, which are capable of degrading tetracycline antibiotics. From soil functional metagenomic selections, we discovered nine genes that confer high-level tetracycline resistance by enzymatic inactivation. We also demonstrate that a tenth enzyme, an uncharacterized homolog in the human pathogen Legionella longbeachae, similarly inactivates tetracycline. These enzymes catalyze the oxidation of tetracyclines in vitro both by known mechanisms and via previously undescribed activity. Tetracycline inactivation genes were identified in diverse soil types, encompass substantial sequence diversity, and are adjacent to genes implicated in horizontal gene transfer. Because tetracycline inactivation is scarcely observed in hospitals, these enzymes may fill an empty niche in pathogenic organisms, and should therefore be monitored for their dissemination potential into the clinic.

Plasticity, dynamics, and inhibition of emerging tetracycline resistance enzymes

“…3), and found to convert chlortetracycline ( m/z 479) to an oxidation product with an m/z of 467. This is in contrast to the m/z 461 monooxygenation product observed for tetracycline 28 , consistent with an alternate binding mode for chlortetracycline. To further characterize this product, reactions were subjected to high resolution mass spectrometry (Fig.…”

“…We reasoned that a similar strategy might be useful to counteract tetracycline resistance by inactivation, and therefore sought to identify small molecule inhibitors of these enzymes. Previously, we observed that anhydrotetracycline, a key biosynthetic precursor 41 and degradation product 42 of tetracycline with poor antibiotic activity was not degraded by Tet(47-56) 28 . None the less, it is known to be an effector of tetracycline producers and tetracycline-resistant bacteria by inducing expression of energetically expensive tetracycline efflux pumps, permitting tetracycline producers to survive and selecting against tetracycline resistance 43 .…”

“…We observed that the soil-derived human pathogen Legionella longbeachae , the causative agent of Pontiac Fever and Legionnaires' Disease 32,33 encodes a homolog to the tetracycline destructases, termed tet (56). Like the other tetracycline destructases, Tet(56) is able to inactivate tetracycline in vitro and expression of tet (56) in E. coli confers high-level tetracycline resistance 28 . To confirm that tet (56) is a functional resistance determinant in L. longbeachae , we deleted the gene and examined the strain for changes in drug sensitivity.…”

“…Tetracycline inactivation by Tet(X) occurs via catalysis at C11a resulting in a product of m/z 461 28 . Because chlortetracycline binds Tet(50) in an alternative mode that positions C11a far away from the reactive flavin peroxide moiety, we sought to characterize the degradation product to establish substrate hydroxylation.…”

“…We recently identified a new family of tetracycline-inactivating enzymes through functional metagenomic selections for tetracycline resistance from 18 grassland and agricultural soils 9 . We showed that these nine proteins, Tet(47-55), were able to enzymatically inactivate tetracycline, resulting in 16-64 fold increases in minimum inhibitory concentration (MIC) 28 when expressed in E. coli .…”