title: Purification and characterization of a new β-lactamase OXA-205 from Pseudomonas aeruginosa.
abstract: BACKGROUND
  We have identified a novel class 1 integron (1503 bp), named In671 in a clinical Pseudomonas aeruginosa isolate. Integron sequence analysis revealed two gene cassettes, one coding for a new OXA-type β-lactamase designated as OXA-205 and the other coding for the aadB gene that is responsible for aminoglycoside resistance. The 266 amino acid sequence of OXA-205 revealed that this β-lactamase belongs to the Ambler class D showing highest sequence homology to the OXA-2 sub-lineage. Our objective was to purify and characterize β-lactamase OXA-205.
  
  
  METHODS
  Escherichia coli cells were transformed with a plasmid containing cloned bla OXA-205 gene from P. aeruginosa. Purification of overproduced OXA-205 consisted of a single ion-exchange chromatography step. SDS-PAGE and isoelectric focusing were performed to determine the molecular mass and pI, respectively. Size-exclusion chromatography was undertaken to determine the OXA-205 oligomerization state. Substrate hydrolysis reactions were employed to assess enzyme kinetic parameters.
  
  
  RESULTS
  Purification of OXA-205 yielded the enzyme with >95 % purity (as verified by SDS-PAGE). Approximate yield of the protein was estimated to be 20 mg per liter of culture. OXA-205 had a pI at 8.1, molecular mass of 26 kDa and a monomeric native structure. Kinetic analysis revealed that OXA-205 hydrolyzed narrow spectrum substrates, including ampicillin, carbenicillin, oxacillin, penicillin G, cefazolin and cefuroxime. Additionally, we observed a substrate inhibition profile towards carbenicillin and oxacillin, but not with ampicillin or penicillin G. Our results also show that OXA-205 conferred unusually high (among class D β-lactamases) resistance towards inhibition by NaCl.
  
  
  CONCLUSIONS
  OXA-205 can be considered a narrow spectrum monomeric β-lactamase that demonstrates unusually high resistance profile towards inhibition by NaCl.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Natural Sciences, Vilnius University, M. K. Čiurlionio 21/27, 03101, Vilnius, Lithuania. renatas.krasauskas@gf.vu.lt.
authors.forename: R
authors.initials: R
authors.lastname: Krasauskas
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Natural Sciences, Vilnius University, M. K. Čiurlionio 21/27, 03101, Vilnius, Lithuania. danute.labeikyte@gf.vu.lt.
authors.forename: D
authors.initials: D
authors.lastname: Labeikytė
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Natural Sciences, Vilnius University, M. K. Čiurlionio 21/27, 03101, Vilnius, Lithuania. arvydas.markuckas@gf.vu.lt.
authors.forename: A
authors.initials: A
authors.lastname: Markuckas
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Natural Sciences, Vilnius University, M. K. Čiurlionio 21/27, 03101, Vilnius, Lithuania. justas.povilonis@gmail.com.|Thermo Fisher Scientific Baltics, V. A. Graičiūno. 8, 02241, Vilnius, Lithuania. justas.povilonis@gmail.com.
authors.forename: J
authors.initials: J
authors.lastname: Povilonis
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Natural Sciences, Vilnius University, M. K. Čiurlionio 21/27, 03101, Vilnius, Lithuania. julija.armalyte@gf.vu.lt.
authors.forename: J
authors.initials: J
authors.lastname: Armalytė
authors.affiliation: Institute of Microbiology and Virology, Lithuanian University of Health Sciences, Tilžės 18, 47181, Kaunas, Lithuania. ritaplanc@mail.lt.
authors.forename: R
authors.initials: R
authors.lastname: Plančiūnienė
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Natural Sciences, Vilnius University, M. K. Čiurlionio 21/27, 03101, Vilnius, Lithuania. kavaliauskaspov@gmail.com.
authors.forename: P
authors.initials: P
authors.lastname: Kavaliauskas
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Natural Sciences, Vilnius University, M. K. Čiurlionio 21/27, 03101, Vilnius, Lithuania. edita.suziedeliene@gf.vu.lt.
authors.forename: E
authors.initials: E
authors.lastname: Sužiedėlienė
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D006868:Hydrolysis; D032023:Integrons; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism
chemical_list: D047090:beta-Lactams; D001618:beta-Lactamases