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Structure of PBP-A from Thermosynechococcus elongatus, a penicillin-binding protein closely related to class A beta-lactamases.

Urbach C, Evrard C, Pudzaitis V, Fastrez J, Soumillion P, Declercq JP
J Mol Biol 386 109-20 (2009)
Related entries: 2j7v, 2j8y, 2jbf

Cited: 11 times
EuropePMC logo PMID: 19100272

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.

Articles - 2j9o mentioned but not cited (2)

  1. An automated flow for directed evolution based on detection of promiscuous scaffolds using spatial and electrostatic properties of catalytic residues. Chakraborty S. PLoS ONE 7 e40408 (2012)
  2. Prediction of Long Loops with Embedded Secondary Structure using the Protein Local Optimization Program. Miller EB, Murrett CS, Zhu K, Zhao S, Goldfeld DA, Bylund JH, Friesner RA. J Chem Theory Comput 9 1846-4864 (2013)


Reviews citing this publication (1)

  1. Epidemiological expansion, structural studies, and clinical challenges of new β-lactamases from gram-negative bacteria. Bush K, Fisher JF. Annu. Rev. Microbiol. 65 455-478 (2011)

Articles citing this publication (8)

  1. Crystal structures of penicillin-binding proteins 4 and 5 from Haemophilus influenzae. Kawai F, Clarke TB, Roper DI, Han GJ, Hwang KY, Unzai S, Obayashi E, Park SY, Tame JR. J. Mol. Biol. 396 634-645 (2010)
  2. Inhibition of a cold-active alkaline phosphatase by imipenem revealed by in silico modeling of metallo-β-lactamase active sites. Chakraborty S, Asgeirsson B, Minda R, Salaye L, Frère JM, Rao BJ. FEBS Lett. 586 3710-3715 (2012)
  3. Classification of Beta-lactamases and penicillin binding proteins using ligand-centric network models. Öztürk H, Ozkirimli E, Özgür A. PLoS ONE 10 e0117874 (2015)
  4. Novel peptide inhibiting both TEM-1 β-lactamase and penicillin-binding proteins. Phichith D, Bun S, Padiolleau-Lefevre S, Guellier A, Banh S, Galleni M, Frere JM, Thomas D, Friboulet A, Avalle B. FEBS J. 277 4965-4972 (2010)
  5. Structural phylogeny by profile extraction and multiple superimposition using electrostatic congruence as a discriminator. Chakraborty S, Rao BJ, Baker N, Asgeirsson B. Intrinsically Disord Proteins 1 e25463 (2013)
  6. Structural and mechanistic studies of the orf12 gene product from the clavulanic acid biosynthesis pathway. Valegård K, Iqbal A, Kershaw NJ, Ivison D, Généreux C, Dubus A, Blikstad C, Demetriades M, Hopkinson RJ, Lloyd AJ, Roper DI, Schofield CJ, Andersson I, McDonough MA. Acta Crystallogr. D Biol. Crystallogr. 69 1567-1579 (2013)
  7. Systematic Identification and Classification of β-Lactamases Based on Sequence Similarity Criteria: β-Lactamase Annotation. Silveira MC, Azevedo da Silva R, Faria da Mota F, Catanho M, Jardim R, R Guimarães AC, de Miranda AB. Evol. Bioinform. Online 14 1176934318797351 (2018)
  8. β-Lactamases Evolve against Antibiotics by Acquiring Large Active-Site Electric Fields. Ji Z, Boxer SG. J Am Chem Soc 144 22289-22294 (2022)


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