Jacques Fastrez

Phage display as a tool for the directed evolution of enzymes

Since its introduction in 1985, phage display has had a tremendous impact on the discovery of peptides that bind to a variety of receptors, the generation of binding sites within predefined scaffolds, and the creation of high-affinity antibodies without immunization. Its application to enzymology has required the development of techniques that couple enzymatic activity to selection protocols based on affinity chromatography. Here, we describe both indirect methods, using transition-state analogues and suicide substrates, and direct methods, using the ability of active phage-enzymes to transform substrate into product. The methods have been applied to large libraries for mechanistic-based studies and to generate variants with new or improved properties. In addition, such techniques have been successfully used to select catalytic antibodies and improve their catalytic efficiency.

Transforming Carbonic Anhydrase into Epoxide Synthase by Metal Exchange

An attractive hypothesis in enzyme evolution relies on the promiscuous activities that already exist in enzymes, which can be used as pipelines for the selection of new catalytic activities. These new activities are usually low, but can serve as starting points in Darwinian evolution. In metalloenzymes, the diversity of promiscuous activity is increased by the variety of metallic ions that can be incorporated in the active site and can catalyse a wide range of chemical transformations. As an example, the iron-containing rubredoxin oxidase and zinc-b-lactamase are two phylogenetically related enzymes that catalyse oxidation and hydrolysis reactions with unrelated mechanisms. This principle could be applicable in the field of directed evolution as a way to discover primitive artificial metalloenzymes that might catalyse challenging asymmetric transformations. In this work, we replaced the zinc ion of natural carbonic anhydrase with manganese and showed that the resulting product can catalyse the enantioselective epoxidation of styrene. Protein modification with cofactors or metal-containing complexes is an approach that has been widely used for the generation of new catalytic activities, but few examples in the field of asymmetric catalysis have yet appeared. The very first was reported in 1978 by Wilson and Whitesides, on the incorporation of a biotinylated achiral rhodium diphosphine complex in avidin for the hydrogenation of dehydroamino acids; this concept has recently been improved through combination of structural variants of the biotinylated complex with several wild-type and mutant proteins. A similar noncovalent approach was used for the enantioselective oxidation of sulfides after incorporation of a vanadate ion into a hydrolase ACHTUNGTRENNUNGhomologous to vanadium-dependent peroxidases or of an ACHTUNGTRENNUNGachiral chromium salophen complex into apomyoglobin. Covalent grafting of achiral organometallic complexes has also been used as a complementary approach, with attachment of a copper phenanthroline complex to the adipocyte lipid-binding protein affording enantioselective hydrolysis of esters and amides. More recently, the dual anchoring of an achiral manganese salen complex in apomyoglobin for asymmetric sulfoxidation has been reported. In all these approaches the metallic ion is part of a larger achiral complex or ion and the chiral environment is provided by the protein. Our approach consists of incorporating only the catalytic metal ion in a protein featuring a suitable site for coordination, taking advantage of an attractive recently reported procedure for the manganese-catalysed epoxidation of alkenes. This method uses hydrogen peroxide in hydrogen carbonate buffer, and the authors postulated the formation of peroxymonocarbonate—HCO4 —as the actual oxygen-transfer reagent. We selected carbonic anhydrase as a suitable candidate for hosting the manganese ion in its active site because the active species of the epoxidation mechanism and that of the natural reaction were similar. Indeed, carbonic anhydrase is a zinc-containing enzyme that catalyses the reversible hydration of carbon dioxide by a mechanism involving coordination of hydrogenocarbonate ion to the metallic centre. Furthermore, carbonic anhydrase displays a micromolar affinity for manganese ion. As a hydrophobic cavity close to the active site is responsible for enzyme inhibition by small aromatic compounds such as benzenesulfonamides, we decided to select aromatic alkenes as substrates for first trials. We first evaluated the effect of addition of apoenzyme on the rate of the manganese(II)-catalysed epoxidation of 4-vinylbenzoic acid, as monitored by spectrophotometry. The apoenzyme is capable of inhibiting the epoxidation almost completely, and an apparent dissociation constant of 6.5> 10 m was obtained by fitting the data with a second-order equation (Figure 1). The affinity is about 10 times higher than

CENTA as a Chromogenic Substrate for Studying β-Lactamases

ABSTRACT CENTA, a chromogenic cephalosporin, is readily hydrolyzed by β-lactamases of all classes except for the Aeromonas hydrophila metalloenzyme. Although it cannot practically be used for the detection of β-lactamase-producing strains on agar plates, it should be quite useful for kinetic studies and the detection of the enzymes in crude extracts and chromatographic fractions.

Engineering a regulatable enzyme for homogeneous immunoassays.

We have engineered the phage displayed TEM-1 β-lactamase to generate enzymes that can be used in homogeneous immunoassays because their activity can be modulated by binding to monoclonal antibodies (Mabs) raised against an unrelated protein. Random peptide libraries were genetically inserted into three loops to create hybrid enzymes with binding sites for Mabs. Insertion points were chosen to be close enough to the active site that complex formation could affect the activity. The antibiotic resistance provided by the β-lactamase activity was used to select the clones encoding active enzymes. Biopanning of the active libraries on immobilized Mabs against the prostate specific antigen (PSA) or on streptavidin yielded enzymes with binding sites for these proteins. Their activity could be regulated by Mab or streptavidin binding. The dissociation constants of the complexes are in the 10–9 to 10–6 M range. In a competitive assay, PSA could be detected at a minimal concentration of 10–9 M. The Mabs recognize mimotopes as no sequence similarity was found between inserts in regulated clones and fragments of the PSA sequence. The method can be developed to generate signaling molecules to be used for the detection of analytes in solution without identification of the epitope.

In vivo versus in vitro screening or selection for catalytic activity in enzymes and abzymes

The recent development of catalytic antibodies and the introduction of new techniques to generate huge libraries of random mutants of existing enzymes have created the need for powerful tools for finding in large populations of cells those producing the catalytically most active proteins. Several approaches have been developed and used to reach this goal. The screening techniques aim at easily detecting the clones producing active enzymes or abzymes; the selection techniques are designed to extract these clones from mixtures. These techniques have been applied both in vivo and in vitro. This review describes the advantages and limitations of the various methods in terms of ease of use, sensitivity, and convenience for handling large libraries. Examples are analyzed and tentative rules proposed. These techniques prove to be quite powerful to study the relationship between structure and function and to alter the properties of enzymes.

Phage display of enzymes and in vitro selection for catalytic activity.

Despite recent progress, our understanding of enzymes remains limited: the prediction of the changes that should be introduced to alter their properties or catalytic activities in an expected direction remains difficult. An alternative to rational design is selection of mutants endowed with the anticipated properties from a large collection of possible solutions generated by random mutagenesis. We describe here a new technique of in vitro selection of genes on the basis of the catalytic activity of the encoded enzymes.The gene coding for the enzyme to be engineered is cloned into the genome of a filamentous phage, whereas the enzyme itself is displayed on its surface, creating a phage enzyme. A bifunctional organic label containing a suicide inhibitor of the enzyme and a ligand with high affinity for an immobilized receptor are constructed. On incubation of a mixture of phage enzymes, those phages showing an activity on the inhibitor under the conditions of the experiment are labeled. These phages can be recovered by affinity chromatography.The design of the label and the factors controlling the selectivity of the selection are analyzed. The advantages of the technique and its scope in terms of the enzymes that can be engineered are discussed.

Selection of metalloenzymes by catalytic activity using phage display and catalytic elution.

The metallo‐β‐lactamase βLII from Bacillus cereus 569/H/9 was displayed on the filamentous phage fd. The phage‐bound enzyme fd‐βLII was shown to be active on benzylpenicillin as substrate; it could be inactivated by complexation of the essential zinc(II) ion with EDTA and reactivated by addition of a zinc(II) salt. A selection process was designed to extract active phage‐bound enzymes from libraries of mutants in three steps: 1. inactivation of active phage‐bound enzymes by metal ion complexation, 2. binding to substrate‐coated magnetic beads, 3. release of phages capable of transforming the substrate into product upon zinc salt addition. The selection process was first successfully tested on model mixtures containing fd‐βLII plus either a dummy phage, a phage displaying an inactive mutant of the serine β‐lactamase TEM‐1, or inactive and low‐activity mutants of βLII. The selection was then applied to extract active phage‐bound enzymes from a library of mutants generated by mutagenic polymerase chain reaction (PCR). The activity of the library was shown to increase 60‐fold after two rounds of selection. Eleven clones from the second round were randomly picked for sequencing and to characterize their activity and stability.

Synthesis of new phosphonate inhibitors of serine proteases

Abstract Analogues of phenylalanine and lysine esters were synthesized, and their inhibitory power tested in vitro respectively on chymotrypsin, trypsin and urokinase.

TEM-1 β-lactamase as a scaffold for protein recognition and assay

A large number of different proteins or protein domains have been investigated as possible scaffolds to engineer antibody‐like molecules. We have previously shown that the TEM‐1 β‐lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity. From the libraries that were generated, active enzymes binding with high affinities to monoclonal antibodies raised against prostate‐specific antigen, a protein unrelated to β‐lactamase, could be isolated. Antibody binding was shown to affect markedly the enzyme activity. As a consequence, these enzymes have the potential to be used as signaling molecules in direct or competitive homogeneous immunoassay. Preliminary results showed that β‐lactamase clones binding to streptavidin could also be isolated, indicating that some enzymes in the libraries have the ability to recognize proteins other than antibodies. In this paper, we show that, in addition to β‐lactamases binding to streptavidin, β‐lactamase clones binding to horse spleen ferritin and β‐galactosidase could be isolated. Affinity maturation of a clone binding to ferritin allowed obtaining β‐lactamases with affinities comprised between 10 and 20 nM (Kd) for the protein. Contrary to what was observed for β‐lactamases issued from selections on antibodies, enzyme complexation induced only a modest effect on enzyme activity, in the three cases studied. This kind of enzyme could prove useful in replacement of enzyme‐conjugated antibodies in enzyme‐linked immunosorbant assays (ELISA) or in other applications that use antibodies conjugated to an enzyme.

Engineering Allosteric Regulation into Biological Catalysts

Enzymes and ribozymes constitute two classes of biological catalysts. The activity of many natural enzymes is regulated by the binding of ligands that have different structures than their substrates; these ligands are consequently called allosteric effectors. In most allosteric enzymes, the allosteric binding site lies far away from the active site. This implies that communication pathways must exist between these sites. While mechanisms of allosteric regulation were developed more than forty years ago, they continue to be revisited regularly. The improved understanding of these mechanisms has led in the past two decades to projects to transform several unregulated enzymes into allosterically regulated ones either by rational design or directed evolution techniques. More recently, ribozymes have also been the object of similar successful engineering efforts. In this review, after briefly summarising recent progress in the theories of allosteric regulation, several strategies to engineer allosteric regulations in enzymes and ribozymes are described and compared. These redesigned biological catalysts find applications in a variety of areas.