title: Identification of the enzyme responsible for N-acetylation of norfloxacin by Microbacterium sp. Strain 4N2-2.
abstract: Microbacterium 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.
authors.affiliation: Divisions of Microbiology, U.S. Food and Drug Administration, Jefferson, Arkansas, USA.
authors.forename: Dae-Wi
authors.initials: DW
authors.lastname: Kim
authors.forename: Jinhui
authors.initials: J
authors.lastname: Feng
authors.forename: Huizhong
authors.initials: H
authors.lastname: Chen
authors.forename: Ohgew
authors.initials: O
authors.lastname: Kweon
authors.forename: Yuan
authors.initials: Y
authors.lastname: Gao
authors.forename: Li-Rong
authors.initials: LR
authors.lastname: Yu
authors.forename: Vanessa J
authors.initials: VJ
authors.lastname: Burrowes
authors.forename: John B
authors.initials: JB
authors.lastname: Sutherland
mesh_terms: D000107:Acetylation; D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001665:Binding Sites; D001711:Biotransformation; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D063206:N-Terminal Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009643:Norfloxacin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D062065:Wastewater / Q000382:microbiology
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D005974:Glutamate-Ammonia Ligase; D063206:N-Terminal Acetyltransferases; D009643:Norfloxacin; D011994:Recombinant Proteins; D062065:Wastewater