Cloning of a Chryseobacterium (Flavobacterium) meningosepticum chromosomal gene (blaA(CME)) encoding an extended-spectrum class A beta-lactamase related to the Bacteroides cephalosporinases and the VEB-1 and PER beta-lactamases.
In addition to the BlaB metallo-beta-lactamase, Chryseobacterium (Flavobacterium) meningosepticum CCUG 4310 (NCTC 10585) constitutively produces a 31-kDa active-site serine beta-lactamase, named CME-1, with an alkaline isoelectric pH. The blaA(CME) gene that encodes the latter enzyme was isolated from a genomic library constructed in the Escherichia coli plasmid vector pACYC184 by screening for cefuroxime-resistant clones. Sequence analysis revealed that the CME-1 enzyme is a new class A beta-lactamase structurally divergent from the other members of this class, being most closely related to the VEB-1 (also named CEF-1) and PER beta-lactamases and the Bacteroides chromosomal cephalosporinases. The blaA(CME) determinant is located on the chromosome and exhibits features typical of those of C. meningosepticum resident genes. The CME-1 protein was purified from an E. coli strain that overexpresses the cloned gene via a T7-based expression system by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. Kinetic parameters for several substrates were determined. CME-1 is a clavulanic acid-susceptible extended-spectrum beta-lactamase that hydrolyzes most cephalosporins, penicillins, and monobactams but that does not hydrolyze cephamycins and carbapenems. The enzyme exhibits strikingly different kinetic parameters for different classes of beta-lactams, with both K(m) and k(cat) values much higher for cephalosporins than for penicillins and monobactams. However, the variability of both kinetic parameters resulted in overall similar acylation rates (k(cat)/K(m) ratios) for all types of beta-lactam substrates.
Rossolini
G M
GM
Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università degli Studi di Siena, 53100 Siena, Italy. rossolini@unisi.it
Franceschini
N
N
Lauretti
L
L
Caravelli
B
B
Riccio
M L
ML
Galleni
M
M
Frère
J M
JM
Amicosante
G
G
D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping; D001618:beta-Lactamases / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification
D001618:beta-Lactamases