Role of changes in the L3 loop of the active site in the evolution of enzymatic activity of VIM-type metallo-beta-lactamases.
OBJECTIVE
The new metallo-beta-lactamase VIM-13 has been recently characterized. In comparison with the VIM-1 enzyme, VIM-13 showed 19 amino acid differences, 2 of which were located in the active site centre. The main objective of the present study was to assess whether differences between VIM-1 and VIM-13 beta-lactamases in the active site, at His224Leu and Ser228Arg, are necessary and sufficient to explain the microbiological and biochemical differences between the two enzymes.


METHODS
Single mutants VIM-13 (Leu224His) and VIM-13 (Arg228Ser) and double mutant VIM-13 (Leu224His, Arg228Ser) were created by site-directed mutagenesis with the bla(VIM-13) gene as template. VIM-1, VIM-13 and VIM-13 (Leu224His, Arg228Ser) were purified by affinity chromatography, and kinetic parameters for these enzymes were obtained with ceftazidime, cefepime and ampicillin.


RESULTS
Ceftazidime and cefepime MICs (mg/L) for Escherichia coli TG1 expressing VIM-1, VIM-13, VIM-13 (Leu224His), VIM-13 (Arg228Ser) and VIM-13 (Leu224His, Arg228Ser) were >256 and 64, 6 and 4, 8 and 1, >256 and 8, and >256 and 48, respectively. VIM-1, VIM-13 and VIM-13 (Leu224His, Arg228Ser) revealed k(cat)/K(m) values (M(-1)s(-1)) for ceftazidime of 3.7 E(4), 1.9 E(4) and 10 E(4), respectively, and revealed k(cat)/K(m) values for cefepime of 3.5 E(5), 3 E(4) and 1.5 E(5), respectively.


CONCLUSIONS
Overall, the results showed that the two residues located in the L3 loop are sufficient to confer the substrate specificity of each enzyme, thus highlighting the importance of the L3 loop of the active site in the evolution of VIM-type metallo-beta-lactamases.
Merino
María
M
Servicio de Microbiología-INIBIC, Complejo Hospitalario Universitario La Coruña, 15006 La Coruña, Spain.
Pérez-Llarena
Francisco J
FJ
Kerff
Frederic
F
Poza
Margarita
M
Mallo
Susana
S
Rumbo-Feal
Soraya
S
Beceiro
Alejandro
A
Juan
Carlos
C
Oliver
Antonio
A
Bou
Germán
G
D019943:Amino Acid Substitution; D000667:Ampicillin / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000378:metabolism; D020134:Catalytic Domain; D000077723:Cefepime; D002442:Ceftazidime / Q000378:metabolism; D002511:Cephalosporins / Q000378:metabolism; D002846:Chromatography, Affinity; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D019143:Evolution, Molecular; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D004269:DNA, Bacterial; D050505:Mutant Proteins; D000667:Ampicillin; D000077723:Cefepime; D002442:Ceftazidime; D001618:beta-Lactamases