2J9O

Structure of PBP-A, L158E mutant

Summary for 2J9O

• Entry DOI: 10.2210/pdb2j9o/pdb
• Related: 2J7V 2J8Y
• Descriptor: TLL2115 PROTEIN, GLYCEROL (3 entities in total)
• Functional Keywords: penicillin-binding protein, hydrolase, lactamase, thioesterase, dd-peptidase
• Biological source: SYNECHOCOCCUS ELONGATUS
• Total number of polymer chains: 4
• Total formula weight: 131557.36

Authors

Evrard, C.,Declercq, J.P. (deposition date: 2006-11-14, release date: 2007-12-11, Last modification date: 2024-05-08)

Primary citation

Urbach, C.,Evrard, C.,Pudzaitis, V.,Fastrez, J.,Soumillion, P.,Declercq, J.P.
Structure of Pbp-A from Thermosynechococcus Elongatus, a Penicillin-Binding Protein Closely Related to Class a Beta-Lactamases.
J.Mol.Biol., 386:109-, 2009

PubMed 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.

PubMed: 19100272
DOI: 10.1016/J.JMB.2008.12.001

Experimental method

X-RAY DIFFRACTION (1.5 Å)