title: Structure of PBP-A from Thermosynechococcus elongatus, a penicillin-binding protein closely related to class A beta-lactamases.
abstract: 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 beta-lactamases are phylogenetically linked but feature large differences of reactivity towards their respective substrates. In particular, while beta-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 beta-lactamases but having a six-amino-acid deletion in the conserved Omega-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 beta-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 Omega-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 A resolution and of L158E mutant at 1.5 A 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 beta-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.
authors.affiliation: Laboratoire d'Ingénierie des Protéines et des Peptides, Institut des Sciences de la Vie, Université catholique de Louvain, Place Croix du Sud 4-5, bte3, 1348 Louvain la-Neuve, Belgium.
authors.forename: Carole
authors.initials: C
authors.lastname: Urbach
authors.forename: Christine
authors.initials: C
authors.lastname: Evrard
authors.forename: Vaidas
authors.initials: V
authors.lastname: Pudzaitis
authors.forename: Jacques
authors.initials: J
authors.lastname: Fastrez
authors.forename: Patrice
authors.initials: P
authors.lastname: Soumillion
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Declercq
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000378:metabolism*; D019143:Evolution, Molecular; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046915:Penicillin-Binding Proteins / Q000737:chemistry*; D010406:Penicillins / Q000378:metabolism; D011487:Protein Conformation; D040681:Structural Homology, Protein; D001618:beta-Lactamases / Q000737:chemistry / Q000145:classification
chemical_list: D046915:Penicillin-Binding Proteins; D010406:Penicillins; D001618:beta-Lactamases