Active TEM‐1 β‐lactamase mutants with random peptides inserted in three contiguous surface loops

Abstract

Engineering of alternative binding sites on the surface of an enzyme while preserving the enzymatic activity would offer new opportunities for controlling the activity by binding of non-natural ligands. Loops and turns are the natural substructures in which binding sites might be engineered with this purpose. We have genetically inserted random peptide sequences into three relatively rigid and contiguous loops of the TEM-1 b-lactamase and assessed the tolerance to insertion by the percentage of active mutants. Our results indicate that tolerance to insertion could not be correlated to tolerance to mutagenesis. A turn between two b-strands bordering the active site was observed to be tolerant to random mutagenesis but not to insertions. Two rigid loops comprising rather well-conserved amino acid residues tolerated insertions, although with some constraints. Insertions between the N-terminal helix and the first b-strand generated active libraries if cysteine residues were included at both ends of the insert, suggesting the requirement for a stabilizing disulfide bridge. Random sequences were relatively well accommodated within the loop connecting the final b-strand to the C-terminal helix, particularly if the wild-type residue was retained at one of the loops' end. This suggests two strategies for increasing the percentage of active mutants in insertion libraries. The amino acid distribution in the engineered loops was analyzed and found to be less biased against hydrophobic residues than in natural mediumsized loops. The combination of these activity-selected libraries generated a huge library containing active hybrid enzymes with all three loops modified.

Deactivation of TEM‐1 β‐Lactamase Investigated by Isothermal Batch and Non‐Isothermal Continuous Enzyme Membrane Reactor Methods

“…[22] The predicted Δ G cat was slightly lower in the three-state batch-mode Equilibrium Model, at 53.6 kJ mol −1 , corresponding to a turnover number of 2550 s −1 at 25 °C. The discrepancy between these two values could be a consequence of experimental error associated with the Bradford assay in determining the initial enzyme concentration, or it may be due to determinations of k cat having been made under conditions for which not all of the enzyme is active.…”

Engineering an Allosteric Binding Site for Aminoglycosides into TEM1‐β‐Lactamase

“…The lack of any insertion in loop L3 might have resulted from its rare occurrence (~7 %) in libraries selected for catalytic activity. [24] The backbone amide chemical-shift perturbations observed in the NMR spectra on addition of KanA to BlaKr in MES buffer ( Figure 5 A) could have arisen from KanA binding, MES expulsion or a combination of both. A plot of the difference between the BlaKr spectra in phosphate or MES buffer versus the perturbations induced by Kan-binding in MES sheds some light on this question (Figure 6 A).…”

“…In this library, random peptide sequences had been inserted in loops between the N-and C-terminal helices and their adjacent b-strands, and three residues in a loop connecting two b-strands bordering the active site had been randomized. [24] It was subjected to several rounds of biopanning on immobilized biotinylated kanamycin A (KanA, Scheme 1), then to screening for the effects of KanA on activity. Error-prone PCR was then applied to improve these effects.…”

“…It was purified by following published procedures. [24,34,42] Uniformly labelled 15 N and 13 C proteins were produced in M9 minimal medium with 15 NH 4 C1 (99 % pure, Cambridge Isotope Laboratories) and 13 C 6 -d-glucose (99 % pure, CortecNet, Paris) as sole nitrogen and sugar sources. Protein concentrations were determined from UV/ Vis spectra by using a calculated [43] value of the extinction coefficient, e 280 = 28.21 mm À1 cm À1 .…”

A General Method for Insertion of Functional Proteins within Proteins via Combinatorial Selection of Permissive Junctions

“…Thus, in principle, one should be able to construct new proteins by inserting some of these secondary structure elements (guests) into a new protein partner (host). Indeed, in early pioneering studies, different guests have been successfully inserted into host proteins, including short peptides and cytochrome b 562 into b-lactamase (Edwards et al, 2008;Mathonet et al, 2006), b-lactamase into maltodextrin-binding protein (Betton et al, 1997), calmodulin into GFP (Baird et al, 1999), 1,4-b-xylanase into 1,3-1,4-b-glucanase (Ay et al, 1998), and, most recently, different hormones into bovine and humanized antibodies Zhang et al, 2015;Zhang et al, 2013b;Zhang et al, 2013c). However, these studies were design based, whereas here we present a selection-based method that is arguably much more powerful and general.…”