[en] The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) beta-lactamase from Aeromonas hydrophila is a Zn(2+)-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His(118), Asp(120), His(196) and His(263)) and in substrate specificity (Val(67), Thr(157), Lys(224) and Lys(226)), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp(120) and His(263), and thus is located in the 'cysteine' zinc-binding site. His(118) and His(196) residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val(67) plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val(67) also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys(224) in the binding of substrate. Lys(226) is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Bebrone, Carine ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Anne, Christine ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Kerff, Frédéric ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Garau, Gianpiero
De Vriendt, Kris
Lantin, Raphael; Université de Liège - ULiège > Centre d'ingénierie des protéines
Devreese, Bart
Van Beeumen, Jozef
Dideberg, Otto
Frère, Jean-Marie ; Université de Liège - ULiège > Département des sciences de la vie > Département des sciences de la vie
Galleni, Moreno ; Université de Liège - ULiège > Département des sciences de la vie > Macromolécules biologiques
Language :
English
Title :
Mutational analysis of the zinc- and substrate-binding sites in the CphA metallo-beta-lactamase from Aeromonas hydrophila.
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