Characterization of OXA-29 from Legionella (Fluoribacter) gormanii: molecular class D beta-lactamase with unusual properties.
A class D beta-lactamase determinant was isolated from the genome of Legionella (Fluoribacter) gormanii ATCC 33297(T). The enzyme, named OXA-29, is quite divergent from other class D beta-lactamases, being more similar (33 to 43% amino acid identity) to those of groups III (OXA-1) and IV (OXA-9, OXA-12, OXA-18, and OXA-22) than to other class D enzymes (21 to 24% sequence identity). Phylogenetic analysis confirmed the closer ancestry of OXA-29 with members of the former groups. The OXA-29 enzyme was purified from an Escherichia coli strain overexpressing the gene via a T7-based expression system by a single ion-exchange chromatography step on S-Sepharose. The mature enzyme consists of a 28.5-kDa polypeptide and exhibits an isoelectric pH of >9. Analysis of the kinetic parameters of OXA-29 revealed efficient activity (k(cat)/K(m) ratios of >10(5) M(-1) x s(-1)) for several penam compounds (oxacillin, methicillin, penicillin G, ampicillin, carbenicillin, and piperacillin) and also for cefazolin and nitrocefin. Oxyimino cephalosporins and aztreonam were also hydrolyzed, although less efficiently (k(cat)/K(m) ratios of around 10(3) M(-1) x s(-1)). Carbapenems were neither hydrolyzed nor inhibitory. OXA-29 was inhibited by BRL 42715 (50% inhibitory concentration [IC(50)], 0.44 microM) and by tazobactam (IC(50), 3.2 microM), but not by clavulanate. It was also unusually resistant to chloride ions (IC(50), >100 mM). Unlike OXA-10, OXA-29 was apparently found as a dimer both in diluted solutions and in the presence of EDTA. Its activity was either unaffected or inhibited by divalent cations. OXA-29 is a new class D beta-lactamase that exhibits some unusual properties likely reflecting original structural and mechanistic features.
Franceschini
N
N
Dipartimento di Scienze e Tecnologie Biomediche, Università di L'Aquila, I-67100 L'Aquila, Italy.
Boschi
L
L
Pollini
S
S
Herman
R
R
Perilli
M
M
Galleni
M
M
Frère
J M
JM
Amicosante
G
G
Rossolini
G M
GM
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins; D002614:Chelating Agents / Q000494:pharmacology; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D007875:Legionella / Q000201:enzymology* / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D021241:Spectrometry, Mass, Electrospray Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002614:Chelating Agents; D004274:DNA, Recombinant; D013438:Sulfhydryl Compounds; D004492:Edetic Acid; C445687:OXA-29 protein, Fluoribacter gormanii; D001618:beta-Lactamases