Identification of the Enzyme Responsible forN-Acetylation of Norfloxacin by Microbacterium sp. Strain 4N2-2

Abstract

bMicrobacterium sp. 4N2-2, isolated from a wastewater treatment plant, converts the antibacterial fluoroquinolone norfloxacin to N-acetylnorfloxacin and three other metabolites. Because N-acetylation results in loss of antibacterial activity, identification of the enzyme responsible is important for understanding fluoroquinolone resistance. The enzyme was identified as glutamine synthetase (GS); N-acetylnorfloxacin was produced only under conditions associated with GS expression. The GS gene (glnA) was cloned, and the protein (53 kDa) was heterologously expressed and isolated. Optimal conditions and biochemical properties (K m and V max ) of purified GS were characterized; the purified enzyme was inhibited by Mn 2؉ , Mg 2؉ , ATP, and ADP. The contribution of GS to norfloxacin resistance was shown by using a norfloxacin-sensitive Escherichia coli strain carrying glnA derived from Microbacterium sp. 4N2-2. The GS of Microbacterium sp. 4N2-2 was shown to act as an N-acetyltransferase for norfloxacin, which produced low-level norfloxacin resistance. Structural and docking analysis identified potential binding sites for norfloxacin at the ADP binding site and for acetyl coenzyme A (acetyl-CoA) at a cleft in GS. The results suggest that environmental bacteria whose enzymes modify fluoroquinolones may be able to survive in the presence of low fluoroquinolone concentrations. Fluoroquinolones are widely used as human and veterinary antimicrobial agents (1). Because their persistence in the environment (2) may act as a selective pressure for naïve strains to acquire resistance (3), an understanding of the fate of these drugs should help to prevent drug resistance. The principal resistance mechanisms for fluoroquinolones include the following: (i) mutation of genes for the drug targets (GyrA, GyrB, and ParC) (4-6), (ii) mimicking the drug targets (QnrA, QnrB, and QnrS) (7-9), (iii) reduction of drug accumulation in the cells (AcrAB-TolC and QepA) (4, 10, 11), and (iv) enzymatic modification of the drug [N-acetylation by AAC(6=)-Ib-cr] (12-14).In some strains of Escherichia coli, a mutated aminoglycoside acetyltransferase, AAC(6=)-Ib-cr, acetylates fluoroquinolones at the N-terminal of the piperazine ring (12-14) and enhances bacterial resistance to these drugs (14). Production of N-acetylnorfloxacin and N-nitrosonorfloxacin by environmental Mycobacterium sp. strains has also been found, but the enzymes responsible for modifications are unknown (15) and the aac(6=)-Ib-cr variant gene has not been detected in these strains (12).A norfloxacin-modifying bacterium, Microbacterium sp. strain 4N2-2, was isolated from wastewater (16). Some strains of this genus have been isolated from human clinical specimens, and the majority of them have shown fluoroquinolone resistance (17). Microbacterium sp. 4N2-2 transforms norfloxacin into four different metabolites, including N-acetylnorfloxacin, and cell extracts of this strain catalyze the N-acetylation of norfloxacin (16). NAcetylation is enhanced by Casamino Acids and inhibited by ammo...

Unusual and Unconsidered Mechanisms of Bacterial Resilience and Resistance to Quinolones

“…For instance, Kim et al described four different norfloxacin modification routes in Microbacterium: hydroxylation, oxidative defluorination, desethylation and N-acetylation [165]. In 2013, the same group described that norfloxacin N-acetylation in Microbacterium was related to the action of a glutamine synthetase encoded in the gene glnA (GenBank access: JX901058), confirm-ing this finding by cloning and expressing this gene in E. coli, and observing both the ability of the enzyme cloned in E. coli to modify norfloxacin and its effect on ciprofloxacin susceptibility [168] (Figure 5). modification (i.e., AAC(6′)Ib-cr, CrpP) has also been reported, with descriptions of danofloxacin modifications by P. aeruginosa [167].…”

“…Thus, an inverse relationship has been observed between the levels of expression In the cells with the glnA insert, the reduced diameter of the clear zones of inhibition due to induction of glutamine synthetase was statistically significant (p < 0.05). Reproduced from reference [168] with the permission of Elsevier.…”

Dynamic microfluidic single-cell screening identifies pheno-tuning compounds to potentiate tuberculosis therapy

“…No reuse allowed without permission. Moreover, other N-acetyltransferases in the mycobacterial cell could be responsible for this modification 42 .…”

“…On the other hand, bioreduction of nitroaromatic compounds has been implicated in bacterial cell poisoning 51 . Thus, the nitro group of M06 may also act as a functional electron trap, contributing to the redox toxicity of this compound in the mycobacterial cell, which can carry out several bioreactions on this PTC 40,41,42 . Deregulation of redox balance was also shown to alter the activity of human NAT, via reversible oxidation of the catalytic cysteine 52 .…”

Isolation and characterization of a marine bacterium Vibrio diabolicus strain L2-2 capable of biotransforming sulfonamides

“…Acetylation is an important metabolic pathway for resistance, detoxification, and inactivation for drugs such as aminoglycosides (Soleimani et al, 2018), fluoroquinolone (Kim et al, 2013), streptothricin (Burckhardt and Escalante-Semerena, 2017), and others. NAT has been confirmed to catalyze the acetyl-CoA-dependent N-acetylation of various arylamines and their N-hydroxylated metabolites (WEBER, 1985).…”