Structure of PBP-A from Thermosynechococcus elongatus, a Penicillin-Binding Protein Closely Related to Class A β-Lactamases

JOURNAL OF MOLECULAR BIOLOGY

卷:386

Article

Urbach, Carole¹  Evrard, Christine²  Pudzaitis, Vaidas¹  Fastrez, Jacques¹  Soumillion, Patrice¹  Declercq, Jean-Paul² 

[1] Catholic Univ Louvain, Inst Sci Vie, Lab Ingn Prot & Peptides, B-1348 Louvain, Belgium

[2] Catholic Univ Louvain, Unit Struct Chem, CSTR, B-1348 Louvain, Belgium

关键词: DD-peptidase;    beta-lactamase;    PBP;    carboxypeptidase;    cyanobacteria;   

DOI  :  10.1016/j.jmb.2008.12.001

来源: Elsevier

【 摘 要 】

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, h e 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 angstrom resolution and of L158E mutant at 1.5 angstrom 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. (C) 2008 Elsevier Ltd. All rights reserved.

【 授权许可】

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