Structure of PBP-A from Thermosynechococcus elongatus, a penicillin-binding protein closely related to class A beta-lactamases
- PMID: 19100272
- DOI: 10.1016/j.jmb.2008.12.001
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.
Similar articles
-
Trapping of an acyl-enzyme intermediate in a penicillin-binding protein (PBP)-catalyzed reaction.J Mol Biol. 2008 Feb 15;376(2):405-13. doi: 10.1016/j.jmb.2007.10.066. Epub 2007 Nov 1. J Mol Biol. 2008. PMID: 18155726
-
The D-methyl group in beta-lactamase evolution: evidence from the Y221G and GC1 mutants of the class C beta-lactamase of Enterobacter cloacae P99.Biochemistry. 2005 May 24;44(20):7543-52. doi: 10.1021/bi050136f. Biochemistry. 2005. PMID: 15895997
-
Role of the omega-loop in the activity, substrate specificity, and structure of class A beta-lactamase.Biochemistry. 1998 Mar 10;37(10):3286-96. doi: 10.1021/bi972127f. Biochemistry. 1998. PMID: 9521648
-
Dissecting the catalytic mechanism of a plant beta-D-glucan glucohydrolase through structural biology using inhibitors and substrate analogues.Carbohydr Res. 2007 Sep 3;342(12-13):1613-23. doi: 10.1016/j.carres.2007.05.013. Epub 2007 May 18. Carbohydr Res. 2007. PMID: 17548065 Review.
-
Metallo-beta-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily.Biochem Pharmacol. 2007 Dec 15;74(12):1686-701. doi: 10.1016/j.bcp.2007.05.021. Epub 2007 Jun 2. Biochem Pharmacol. 2007. PMID: 17597585 Review.
Cited by
-
β-Lactamases Evolve against Antibiotics by Acquiring Large Active-Site Electric Fields.J Am Chem Soc. 2022 Dec 7;144(48):22289-22294. doi: 10.1021/jacs.2c10791. Epub 2022 Nov 18. J Am Chem Soc. 2022. PMID: 36399691 Free PMC article.
-
Systematic Identification and Classification of β-Lactamases Based on Sequence Similarity Criteria: β-Lactamase Annotation.Evol Bioinform Online. 2018 Sep 10;14:1176934318797351. doi: 10.1177/1176934318797351. eCollection 2018. Evol Bioinform Online. 2018. PMID: 30210232 Free PMC article.
-
Classification of Beta-lactamases and penicillin binding proteins using ligand-centric network models.PLoS One. 2015 Feb 17;10(2):e0117874. doi: 10.1371/journal.pone.0117874. eCollection 2015. PLoS One. 2015. PMID: 25689853 Free PMC article.
-
Structural phylogeny by profile extraction and multiple superimposition using electrostatic congruence as a discriminator.Intrinsically Disord Proteins. 2013;1(1):e25463. doi: 10.4161/idp.25463. Intrinsically Disord Proteins. 2013. PMID: 25364645 Free PMC article.
-
An automated flow for directed evolution based on detection of promiscuous scaffolds using spatial and electrostatic properties of catalytic residues.PLoS One. 2012;7(7):e40408. doi: 10.1371/journal.pone.0040408. Epub 2012 Jul 11. PLoS One. 2012. PMID: 22811760 Free PMC article.
Publication types
MeSH terms
Substances
Associated data
- Actions
- Actions
- Actions
- Actions
LinkOut - more resources
Full Text Sources
Research Materials
Miscellaneous