Patrice Soumillion

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

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.

New generation of amino coumarin methyl sulfonate-based fluorogenic substrates for amidase assays in droplet-based microfluidic applications.

Droplet-based microfluidics is a powerful tool for biology and chemistry as it allows the production and the manipulation of picoliter-size droplets acting as individual reactors. In this format, high-sensitivity assays are typically based on fluorescence, so fluorophore exchange between droplets must be avoided. Fluorogenic substrates based on the coumarin leaving group are widely used to measure a variety of enzymatic activities, but their application in droplet-based microfluidic systems is severely impaired by the fast transport of the fluorescent product between compartments. Here we report the synthesis of new amidase fluorogenic substrates based on 7-aminocoumarin-4-methanesulfonic acid (ACMS), a highly water-soluble dye, and their suitability for droplet-based microfluidics applications. Both substrate and product had the required spectral characteristics and remained confined in droplets from hours to days. As a model experiment, a phenylacetylated ACMS was synthesized and used as a fluorogenic substrate of Escherichia coli penicillin G acylase. Kinetic parameters (k(cat) and K(M)) measured in bulk and in droplets on-chip were very similar, demonstrating the suitability of this synthesis strategy to produce a variety of ACMS-based substrates for assaying amidase activities both in microtiter plate and droplet-based microfluidic formats.

Lactate racemase is a nickel-dependent enzyme activated by a widespread maturation system

Racemases catalyze the inversion of stereochemistry in biological molecules, giving the organism the ability to use both isomers. Among them, lactate racemase remains unexplored due to its intrinsic instability and lack of molecular characterization. Here we determine the genetic basis of lactate racemization in Lactobacillus plantarum. We show that, unexpectedly, the racemase is a nickel-dependent enzyme with a novel α/β fold. In addition, we decipher the process leading to an active enzyme, which involves the activation of the apo-enzyme by a single nickel-containing maturation protein that requires preactivation by two other accessory proteins. Genomic investigations reveal the wide distribution of the lactate racemase system among prokaryotes, showing the high significance of both lactate enantiomers in carbon metabolism. The even broader distribution of the nickel-based maturation system suggests a function beyond activation of the lactate racemase and possibly linked with other undiscovered nickel-dependent 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.

Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge ?

In the European Union, the definition of a GMO is technology-based. This means that a novel organism will be regulated under the GMO regulatory framework only if it has been developed with the use of defined techniques. This approach is now challenged with the emergence of new techniques. In this paper, we describe regulatory and safety issues associated with the use of oligonucleotide-mediated mutagenesis to develop novel organisms. We present scientific arguments for not having organisms developed through this technique fall within the scope of the EU regulation on GMOs. We conclude that any political decision on this issue should be taken on the basis of a broad reflection at EU level, while avoiding discrepancies at international level.

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.

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.

Novel concepts for selection of catalytic activity

The selection of mutant enzymes with novel properties from libraries is emerging as a very powerful strategy for enzyme engineering. The past year has witnessed significant progress on several fronts: new and improved methods have been developed for the creation of libraries and advances have been made in screening and selection techniques. The results achieved demonstrate the enormous potential of the methods and leave questions open for further studies.

Intein-mediated cyclization of randomized peptides in the periplasm of Escherichia coli and their extracellular secretion.

Split-inteins can be used to generate backbone cyclized peptide as a source of new bioactive molecules. In this work we show that cysteine-mediated splicing can be performed in the oxidative environment of the periplasm of Escherichia coli. Cyclization of the TEM-1 beta-lactamase and of small randomized peptides was demonstrated using an artificially permuted version of the DnaB mini-intein from Synechocystis sp. PCC6803 strain fused to a signal sequence. For small peptides, a signal sequence that promotes cotranslational translocation had to be used. Efficient backbone cyclization was observed for more than 50% of combinatorial peptides featuring a fully randomized sequence inserted between a serine and glycine that are necessary for fast splicing. Furthermore, by coexpressing a mutant of the pIV outer membrane pore protein of fd bacteriophage, we showed that peptides can diffuse in the extracellular medium. These results open new routes for searching compounds acting on new targets such as exported and membrane proteins or pathogen microorganisms.