Role of the conserved amino acids of the 'SDN' loop (Ser130, Asp131 and Asn132) in a class A beta-lactamase studied by site-directed mutagenesis.
Ser130, Asp131 and Asn132 ('SDN') are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.
Jacob
F
F
Laboratoire d'Enzymologie, Université de Liège, Belgium.
Joris
B
B
Lepage
S
S
Dusart
J
J
Frère
J M
JM
D000595:Amino Acid Sequence; D001216:Asparagine; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010397:Penicillanic Acid / Q000378:metabolism / Q000031:analogs & derivatives; D012694:Serine; D013302:Streptomyces / Q000201:enzymology; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D001224:Aspartic Acid; D012694:Serine; D001216:Asparagine; D010397:Penicillanic Acid; C031528:6-iodopenicillanic acid; D001618:beta-Lactamases