2JBF: Structure Of Pbp-A, L158e Mutant. Acyl-Enzyme Complex With Penicillin-G

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.
PDB ID: 2JBFDownload
MMDB ID: 62197
PDB Deposition Date: 2006/12/6
Updated in MMDB: 2012/11
Experimental Method:
x-ray diffraction
Resolution: 1.7  Å
Source Organism:
Similar Structures:
Biological Unit for 2JBF: monomeric; determined by author and by software (PISA)
Molecular Components in 2JBF
Label Count Molecule
Protein (1 molecule)
Tll2115 Protein
Molecule annotation
Chemicals (2 molecules)
* Click molecule labels to explore molecular sequence information.

Citing MMDB