Reference : Structure of PBP-A from Thermosynechococcus elongatus, a Penicillin-Binding Protein C...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/64081
Structure of PBP-A from Thermosynechococcus elongatus, a Penicillin-Binding Protein Closely Related to Class A β-Lactamases
English
Urbach, Carole [Université Catholique de Louvain - UCL > > Institut des sciences de la vie - ISV > >]
Evrard, Christine mailto [Université Catholique de Louvain - UCL > Département de Chimie > Unité de Chimie Structurale - CSTR > >]
Pudzaitis, Vaidas [Université Catholique de Louvain - UCL > > Institut des sciences de la vie - ISV > >]
Fastrez, Jacques mailto [Université Catholique de Louvain - UCL > > Institut des sciences de la vie - ISV > >]
Soumillion, Patrice mailto [Université Catholique de Louvain - UCL > > Institut des sciences de la vie - ISV > >]
Declercq, Jean-Paul mailto [Université Catholique de Louvain - UCL > Département de Chimie > Unité de Chimie Structurale - CSTR > >]
2009
Journal of Molecular Biology
Academic Press
386
109-120
Yes (verified by ORBi)
International
0022-2836
1089-8638
London
United Kingdom
[en] DD-peptidase ; β-lactamase ; PBP ; carboxypeptidase ; cyanobacteria
[en] Molecular evolution has always been a subject of discussions, and researchers are interested in understanding how proteins with similar scaffolds can catalyze different reactions. In the superfamily of serine penicillin-recognizing enzymes, D-alanyl-D-alanine peptidases and β-lactamases are phylogenetically linked but feature large differences of reactivity towards their respective substrates. In particular, while β-lactamases hydrolyze penicillins very fast, leading to their inactivation, these molecules inhibit D-alanyl-D-alanine peptidases by forming stable covalent penicilloyl enzymes. In cyanobacteria, we have discovered a new family of penicillin-binding proteins (PBPs) presenting all the sequence features of class A β-lactamases but having a six-amino-acid deletion in the conserved Ω-loop and lacking the essential Glu166 known to be involved in the penicillin hydrolysis mechanism. With the aim of evolving a member of this family into a β-lactamase, PBP-A from Thermosynechococcus elongatus has been chosen because of its thermostability. Based on sequence alignments, introduction of a glutamate in position 158 of the shorter Ω-loop afforded an enzyme with a 50-fold increase in the rate of penicillin hydrolysis. The crystal structures of PBP-A in the free and penicilloylated forms at 1.9 Å resolution and of L158E mutant at 1.5 Å resolution were also solved, giving insights in the catalytic mechanism of the proteins. Since all the active-site elements of PBP-A-L158E, including an essential water molecule, are almost perfectly superimposed with those of a class A β-lactamase such as TEM-1, the question why our mutant is still 5 orders of magnitude less active as a penicillinase remains and our results emphasize how far we are from understanding the secrets of enzymes. Based on the few minor differences between the active sites of PBP-A and TEM-1,mutations were introduced in the L158E enzyme, but while activities on D-Ala-D-Ala mimicking substrates were severely impaired, further improvement in penicillinase activity was
unsuccessful.
http://hdl.handle.net/2268/64081
http://www.elsevier.com/wps/find/journaldescription.cws_home/622890/description#description
Carole Ubrach and Christine Evrard contributed equally to this work

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