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 penicillinrecognizing enzymes, D-alanyl-D-alanine peptidases and β-lacta-mases are phylogenetically linked but feature large differences of reactivity towards their respective substrates. In particular, while β-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 penicillinbinding proteins (PBPs) presenting all the sequence features of class A β-lactamases but having a six-amino-acid deletion in the conserved Ω-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 β-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 Ω-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 Å resolution and of L158E mutant at 1.5 Å 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 β-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.
We have developed an in vitro evolution method for the selection for catalytic activity under the conditions of free intermolecular interaction between the enzyme and a substrate. The destabilized ternary enzyme-mRNA-ribosome complexes generated by a ribosome display of the mutant library are compartmentalized in vitro by forming a water-in-oil emulsion in such a way, that every droplet would on average contain no more than a single complex. After the complex dissociates within the droplet, the released enzyme molecule is free to interact with a substrate under the selection pressure on all its enzymatic properties (substrate binding, product formation, rate acceleration and turnover) simultaneously-an opportunity for the most efficient selection for catalytic activity. By using the M-MuLV reverse transcriptase as a model, we demonstrated the high efficiency of the method selecting for mutants synthesizing cDNA at increased temperature. A slightly modified compartmentalized ribosome display (CRD) could be used for the selection of other enzymes activities (e.g. DNA polymerase, RNA or DNA ligase terminal nucleotidyl transferase activity). Employment of microfluidics technique could broaden the scope of CRD technique furthermore providing an opportunity to select almost any enzyme at single molecule level under desired conditions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.