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336 Hybrid enzymes Pierre Béguin Combining structural elements belonging to different proteins is a powerful method for generating proteins with new properties. Progress based on detailed structural and functional analysis enables a better integration of the elements to be fitted together while preserving or creating functional interactions between them. Addresses Unité de Physiologie Cellulaire, Département des Biotechnologies, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France; e-mail: [email protected] Current Opinion in Biotechnology 1999, 10:336–340 http://biomednet.com/elecref/0958166901000336 © Elsevier Science Ltd ISSN 0958-1669 Abbreviations Inp ice-nucleating protein L-lactate dehydrogenase LDH MDH malate dehydrogenase Introduction Engineering enzymes to improve their properties is one of the obvious goals of biotechnology. In addition, it has also become an essential tool of research for basic protein biochemistry. In vitro DNA synthesis and recombinant DNA technology make it possible to design and produce any kind of polypeptide. Predicting ab initio the structure, let alone the function of a polypeptide of known sequence, however, still lies beyond the current capabilities of computer modelling. Hence, two strategies are left for the creation or improvement of proteins: firstly, the creation of random libraries; and secondly, the engineering of proteins already existing in Nature. Even with the appearance of powerful screening techniques, such as phage display, the former approach is limited to very short segments, as the number of candidates increases exponentially with the number of residues to be varied independently. Thus, the majority of protein engineering relies on the modification of pre-existing proteins. For this reason, recombination of pre-existing elements provides a very powerful tool to generate proteins with new properties. This paper will review recent advances concerning hybrid enzymes that are most likely to become significant for biotechnology. As pointed out by Nixon et al. [1], the elements being exchanged between different proteins may consist of individual residues (point mutations), secondary structure elements, whole subdomains, or whole proteins (leading to fusion proteins). Shorter elements are usually swapped between homologous functional units, with a view to retain the same basic function with modified properties: kinetic parameters, substrate specificity, thermostability, pH optimum, and so on. Another concept lies in the recruiting of two or more functional units to create bi- or multifunctional proteins. Engineering new properties by recombining homologous domains Pairs of enzymes that are sufficiently related lend themselves to the construction of hybrids, either by in vivo recombination [2] or by extension of overlapping PCR fragments in vitro [3]. Series of recombinants are then analyzed in an attempt to identify determinants responsible for parameters such as thermostability or substrate specificity [4,5]. One problem in exchanging large segments of polypeptides lies in the high probability that the delicate network of interactions required for the proper structure and function of the proteins will be perturbed. Thus, as reviewed recently by Nixon et al. [1], most attempts to modify the substrate or cofactor specificity of the enzymes rely on the site-directed mutagenesis of one or a few residues. An interesting concept, however, lies in the exchange of defined structural subdomains, whose proper folding is more likely to be preserved than it would upon shuffling of arbitrary polypeptide segments. For example, serine proteases of the S1 family are composed of two homologous β-barrel subdomains whose interface forms the active-site cleft. The hydrophobic core structures and the catalytic triad residues are conserved, but the surface loops surrounding the active site are variable, which accounts for the diverse substrate specificity and regulatory properties of the enzymes. Thus, S1 serine proteases are good candidates for generating new enzymes by subdomain swapping. Hopfner et al. [6••] constructed a hybrid protease (fXYa) consisting of the amino-terminal subdomain of the coagulation factor Xa (fXa) and of the carboxy-terminal subdomain of trypsin. Three-dimensional structural analysis of the hybrid showed that the hydrophobic core elements of the interface retained their structure with minor adjustments, but surface elements diverged more extensively from the parental structures. Kinetic parameters of fXa, fXYa, and trypsin were tested with a set of synthetic substrates. The recombinant protease was found to possess amidolytic activity in the same range as the parental enzymes. In general, the hybrid protease displayed less pronounced differences in activity with different substrates than the parental enzymes. Catalytic efficiencies were more similar to those found with trypsin, in agreement with the majority of substrate binding interactions occurring with the trypsin moiety of the hybrid. The molecular recognition profile, however, differed significantly from that of trypsin, with a preference inherited from fXa for substrates carrying glycine as the penultimate residue. Engineering bi- or multi-functional proteins Targeting proteins with specific tags Tagging domains can be defined as domains specifying the distribution of a given polypeptide. They are widely used by organisms to assign proteins to specific locations. As Hybrid enzymes Béguin they usually fold independently from the core protein, they are easy to manipulate, and fusion of proteins with specific tagging domains has now become a standard tool for protein engineers. Vectors encoding signal peptides have long been developed to allow secretion in various heterologous hosts, and many are featured in catalogs of manufacturers of molecular biology supplies. Likewise, various commercial kits are now available to fuse polypeptides of interest with an affinity domain (maltose-binding protein, glutathione S-transferase, cellulose- and chitinbinding domains, hexahistidine etc.) that can be easily purified by affinity chromatography. An interesting development in this area is the construction of a tagging vector, also commercially available, in which the carboxyl terminus of the protein of interest is linked to a chitin-binding domain through a protein splicing element (intein) from Saccharomyces cerevisiae [7]. The intein has been modified so that it undergoes self-cleavage at its amino-terminus upon incubating the fusion protein bound to chitin in the presence of dithiothreitol or β-mercaptoethanol, which liberates the free protein. Displaying polypeptide antigens and proteins on the surface of bacteria has been actively pursued in the perspective of developing live vaccines, reactions catalyzed by enzymes immobilized on whole cells, or whole-cell adsorbents. Insertion of peptide epitopes at permissive sites of the Escherichia coli proteins LamB and MalE has been extensively studied by the group of M Hofnung (reviewed in [8]). A carrier polypeptide was constructed by fusing the signal sequence and the first nine amino acids of E. coli major lipoprotein to five transmembrane segments of the outer membrane protein OmpA. This polypeptide was used to for the successful display of several proteins, such as β-lactamase, a bacterial endoglucanase, a cellulose-binding domain, and single-chain variable fragment (scFv) antibodies [9–11]. Permissive integration of foreign polypeptide sequences within the structure of exocellular proteins is no trivial matter, owing to the possible denaturation of the carrier protein. As an alternative, some exocellular proteins lend themselves to constructions in which the foreign polypeptide is fused at one end of the carrier protein. Recently, the ice-nucleating protein (Inp) of Pseudomonas syringae was used to display Bacillus subtilis endoglucanase [12] or Z. mobilis levansucrase [13•] on the cell surface of E. coli. Attachment of Inp to the cell surface appears to be mediated via a glycosyl phosphatidylinositol anchor [14]. A point of interest is that the length of the carrying polypeptide can be modulated by partial or total deletion of the central region of Inp, which consists of highly repeated segments responsible for ice nucleation [12]. Display systems have also been developed based on the use of specific motifs involved in tethering exocellular proteins to the cell surface. One of them is the carboxy-terminal LPXTG (single letter amino acid code, where X can be any amino acid) motif, which anchors many exocellular proteins to the surface of Gram-positive bacteria. Upon secretion, a transamidation occurs at the level of the threonine of the 337 motif, leading to cleavage of the precursor polypeptide and to a peptide linkage with a free amino group of the peptide cross-bridge in the cell wall [15,16]. This system has been used to display fusion antigens, such as an allergen from hornet venom and a malaria blood stage antigen, on the surface of Streptococcus gordonii [17] and Staphylococcus carnosus [18]. Preliminary evidence indicates that it would also work in various lactic acid bacteria [19], which are non-pathogenic and survive passage along an oral route down to the intestine. Another anchoring motif, termed the SLH domain, has been identified in many bacterial paracrystalline surfacelayer proteins (S-layer proteins) and in several cell-bound exocellular enzymes [20–22]. This domain, which consists of a 60-residue segment usually reiterated threefold, appears to bind to a polysaccharide associated with the cell wall [23]. The SLH domains of the S-layer proteins EA1 and Sap of Bacillus anthracis were recently shown to promote in vivo anchoring of B. subtilis levansucrase to the cell surface of B. anthracis [24•]. Fusion polypeptides have also been considered as a method to target proteins to specific animal cells. The construction of toxic fusion proteins attacking specific disease-causing cells has been actively pursued (reviewed in [25]). In addition, the nontoxic carboxy-terminal fragment of tetanus toxin has been fused to β-galactosidase to label motoneurons and connected neurons by retrograde transport [26]. Fusion to superoxide dismutase enabled delivery and internalization of the enzyme by neuronal cells in culture [27]. The latter work opens up the possibility of treating neurological diseases involving free radical damage to neurons, such as amyotrophic lateral sclerosis. It remains to be demonstrated, however, that the superoxide dismutase-tetanus toxoid hybrid actually protects neurons against such damage. Tagging sequences can also be used to address enzymes to specific DNA sequences. For example, Kim et al. [28] have explored the possibility of creating new restriction enzymes by fusing zinc fingers that bind to specific sequences with the cleavage domain of Fok I endonuclease. The results showed that specific cleavage did occur, although the hybrid enzymes were much less active than wild-type Fok I. The cleavage specificity was variable, some zinc fingers yielding more spurious cleavage at secondary sites than others. DNA recognition sites of RNA polymerase have also been manipulated by creating new hybrid subunits. A hybrid σ subunit was constructed by combining the amino-terminal region of the major E. coli σ subunit (σ70) with the carboxy-terminal end of σ32, which recognizes heat shock promoters. The hybrid promoted transcription from a chimeric promoter composed of the σ32 –35 consensus and of the σ70 –10 consensus sequence [29]. Likewise, Mencía et al. [30] made an RNA polymerase responsive to a B. subtilis transcriptional activator by replacing the wild type E. coli α subunit with a hybrid in which the carboxy-terminal domain was replaced by the corresponding domain of the α subunit from B. subtilis. 338 Protein technologies and commercial enzymes timodule complexes involved in polyketide and nonribosomal peptide synthesis, which yield many compounds with antibiotic or pharmacological activity. In both cases, synthesis proceeds by the sequential addition of specific elements, which is performed by specialized modules. Synthetic modules are part of multimodular polypeptides, which themselves form large synthase complexes responsible for synthesis of the full polyketide or peptide chain. Thus, the order and nature of the modules determines the structure of the final product. Shuffling, deleting or modifying specific modules offers the opportunity for generating an almost unlimited variety of compounds, and the field is currently the focus of intense research activity (for reviews see [33,34•]). Figure 1 Catalytic domains Dockerin domains Cohesin domains Current Opinion in Biotechnology Modelling of artificial complexes having a defined stoichiometry and topology based on cohesin and dockerin domains with different recognition specificities. Cohesin domains are linked together in a defined order into a multimodule scaffolding polypeptide. Catalytic domains to be integrated within the complex are linked to appropriate dockerin domains in order to interact with the various cohesin domains of the scaffolding polypeptide. Linker segments are indicated by black bars. A very important development based on a tagging strategy is the two-hybrid system used for the screening of protein–protein interactions. In the original version, protein association is detected by reconstituting an active transcriptional activator in yeast [31]. More recently, a similar system has been developed based on fusions with Bordetella pertussis adenylate cyclase fragments. Association of the fragments generates sufficient cyclase activity to be monitored by the activity of a reporter gene whose expression is dependent on cAMP. The advantage is that the system is functional in E. coli. In addition, because cAMP is diffusible the interacting partners need not be located in the compartment where transcription occurs [32••]. Natural and artificial systems for grouping different enzymes Nature provides many examples of bi- or multifunctional enzyme systems performing consecutive reactions in various metabolic pathways. Adequate spatial arrangement of catalytic subunits plays a critical role by ensuring that reactions occur in the proper order, and by speeding up the process owing to efficient channeling of intermediate products from one reaction center to the next one. Among the most evolved systems are the mul- Bifunctional proteins constructed de novo to perform coupled reactions were investigated in the late 1980s and early 1990s by Mosbach, Bülow and their co-workers [35–37]. Several fusion proteins were tested, including hybrids of β-galactosidase and galactose dehydrogenase [35], galactose dehydrogenase and bacterial luciferase [36], and malate dehydrogenase and citrate synthase [37]. The steady-state activity of the coupled enzyme systems measured for the coupled reactions was increased up to 2–3-fold as compared to the separated enzymes [35], and the pre-steady state lag was reduced up to 4–6-fold [37]. The enhancement of activity observed upon coupling the reaction centers was maximal when the activity of the first enzyme was limiting owing to non-optimal pH conditions or low concentrations of enzyme or substrate [35,36]. Thus, the effect of coupling on activity was not overwhelming, but nonetheless significant. Better results would probably be expected if it were possible to optimize the position of the catalytic centres relative to one another. As opposed to fusion proteins, hybrid multiprotein complexes offer the possibility to assemble several catalytic centres without synthesizing huge polypeptides. In this respect, the cellulolytic complex, or cellulosome, produced by the bacterium Clostridium thermocellum, lends itself to manipulation with remarkable versatility. The cellulosome consists of up to 25 different enzymes that attack cellulose and associated plant cell-wall polysaccharides. Each of the catalytic subunits is anchored by means of a conserved domain, termed the dockerin domain, to a large scaffolding protein termed CipA. CipA itself consists of a set of nine highly similar receptors, termed cohesin domains, which bind the dockerin domains borne by the catalytic subunits (for a review see [38]). Dockerin domains can be fused to foreign polypeptides, mediating the integration of arbitrarily chosen proteins into artificial complexes [39,40]. Cohesin domains comprised in the sequence of CipA have very similar binding specificities, however, precluding the arrangement of the polypeptides to be associated in a chosen order and stoichiometry. A possible solution lies in the construction of artificial scaffolding polypeptides carrying cohesin domains of differing specificity, whose number and order could be chosen arbitrarily. The Hybrid enzymes Béguin polypeptides to be integrated in the complex would accordingly be fused to dockerin domains of appropriate specificity (Figure 1). Indeed, a heterodimer consisting of two cohesin domains with distinct recognition specificities was shown to mediate the formation of complexes integrating two different polypeptides provided with appropriate dockerin domains [41]. The versatility of the system could be further increased by including cohesin and dockerin domains from related organisms, such as Clostridium cellulolyticum, which have been demonstrated to possess a different recognition specificity [42]. Alternatively, new types of dockerin–cohesin interactions could be designed and created by site-directed mutagenesis when the structure of cohesin–dockerin complexes is elucidated. Engineering allosteric interactions between artificially combined domains Most of the bifunctional proteins described above are made up of domains connected side by side, which presumably retain the capacity to fold independently and possess a considerable degree of conformational freedom relative to one another. Although this ensures that the domains are likely to remain functional within the hybrid, it also means that the spatial orientation of their active sites relative to one another is loose and cannot be optimized. Furthermore, the possibility of engineering allosteric interactions would offer interesting opportunities, for example, by modulating the activity of enzymes, which could then act as biosensors. One way to achieve a closer interaction between two domains is to graft one of them at a permissive site within the sequence of the second one. Accordingly, β-lactamase was fused to the maltodextrinbinding protein MalE, either by insertion at various sites within MalE or by fusion at the carboxyl terminus of the polypeptide chain. Insertion after residues 133 and 303, which were known to accommodate small peptides or deletions, resulted in the secretion into the periplasm of fusion proteins capable of transporting maltose and hydrolyzing β-lactams. The same was true of the carboxyterminal fusion, but the internal fusions displayed two additional properties: firstly, they were less sensitive to degradation by endogenous proteases; and secondly, addition of maltose stabilized the activity of the β-lactamase domain against denaturation by urea, indicating a true allosteric interaction between the two domains [43]. The possibility of developing a sensor system based on the modulation of alkaline phosphatase activity was explored by Brennan et al. [44]. Epitopes of HIV type I and of hepatitis C virus were grafted within the sequence of alkaline phosphatase, and the activity of the recombinant enzyme was tested in the presence or absence of antibodies directed against these epitopes. When epitopes were inserted into wild-type alkaline phosphatase, addition of specific antibodies reduced by 14–17% the activity of the hybrids. Antibodies increased the activity of the fusion proteins up to 2–3-fold when the epitopes were inserted 339 into two mutants of alkaline phosphatase that displayed increased flexibility and lower thermal stability. Finally, allosteric activation can also be manipulated in multimeric complexes. For example, the substrate specificity of L-lactate dehydrogenase (LDH), which is a tetrameric enzyme subject to homotropic activation, can be changed into malate dehydrogenase (MDH) by a single site mutation. Unlike LDH, the resulting MDH does not bind low concentrations of oxamate, a non-reactive analog of pyruvate. Fushinobu et al. [45•] constructed and purified various heterotetramers containing both LDH and MDH. In such hybrids, MDH activity was enhanced in the presence of oxamate, which acted like a homotropic allosteric effector by binding to the LDH sites of the complexes. Conclusions Constructing hybrid polypeptides is a major aspect of protein engineering and covers a very wide range of actual or potential applications. So far, the field has been largely concerned with the mere physical connection of protein segments without paying much attention to the spatial integration of the parts composing the fusion polypeptides. This approach brought about significant success, particularly in the case of tagging strategies, for which tagging domains do not normally need to interact specifically with the rest of the protein. It is probable, however, that more sophisticated designs, based on a detailed knowledge of the structure and function of the elements to be combined, will become increasingly popular. They will be required to develop the full potential of hybrid proteins, particularly for constructing integrated multienzyme complexes or for manipulating the regulation of enzyme activity. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1. Nixon AE, Ostermeier M, Benkovic SJ: Hybrid enzymes: manipulating enzyme design. Trends Biotechnol 1998, 16:258-264. 2. Weber H, Weissmann C: Formation of genes coding for hybrid proteins by recombination between related, cloned genes in Escherichia coli. Nucleic Acids Res 1983, 11:5661-5669. 3. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR: Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 1989, 77:61-68. 4. Conrad B, Hoang V, Polley A, Hofemeister J: Hybrid Bacillus amyloliquefaciens X Bacillus licheniformis a-amylases. Construction, properties and sequence determinants. Eur J Biochem 1995, 230:481-490. 5. Auerswald EA, Nägler DK, Gross S, Assfalg-Machleidt I, Stubbs MT, Eckerkorn C, Machleidt W, Fritz H: Hybrids of chicken cystatin with human kininogen domain 2 sequence exhibit novel inhibition of calpain, improved inhibition of actinidin and impaired inhibition of papain, cathepsin L and cathepsin B. Eur J Biochem 1996, 235:534-542. 6. •• Hopfner K-P, Kopetzki E, Kreße G-B, Bode W, Huber R, Engh RA: New enzyme lineages by subdomain shuffling. Proc Natl Acad Sci USA 1998, 95:9813-9818. An elegant study on the feasibility of modifying the specificity of serine proteases by shuffling subdomains previously identified by 3D structure determination. 7. Chong S, Mersha FB, Comb DG, Scott ME, Landry D, Vence LM, Perler FB, Benner J, Kucera RB, Hirvonen CA et al.: Single-column 340 Protein technologies and commercial enzymes purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein-splicing element. Gene 1997, 192:278-281. 8. 9. Leclerc C, Lo-Man R, Charbit A, Martineau P, Clément J-M, Hofnung M: Immunogenicity of viral B- and T-cell epitopes expressed in recombinant bacterial proteins. Int Rev Immunol 1994, 11:123-132. Francisco JA, Earhart CF, Georgiou G: Transport and anchoring of b-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci USA 1992, 89:2713-2717. 10. Francisco JA, Campbell R, Iverson BL, Georgiou G: Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface. Proc Natl Acad Sci USA 1993, 90:10444-10448. 11. Francisco JA, Stathopoulos C, Warren RAJ, Kilburn DG, Georgiou G: Specific adhesion and hydrolysis of cellulose by intact Escherichia coli expressing surface-anchored cellulase or cellulose-binding domains. Bio/Technology 1993, 11:491-495. 12. Jung H-C, Park J-H, Park S-H, Lebeault J-M, Pan J-G: Expression of carboxymethylcellulase on the surface of Escherichia coli using Pseudomonas syringae ice nucleation protein. Enzyme Microb Technol 1998, 22:348-354. 13. Jung H-C, Lebeault J-M, Pan J-G: Surface display of Zymomonas • mobilis levansucrase by using the ice-nucleation protein of Pseudomonas syringae. Nat Biotechnol 1998, 16:576-580. A new, versatile system for decorating the surface of E. coli with foreign polypeptides. 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Kumar A, Grimes B, Logan M, Wedgwood S, Williamson H, Hayward RS: A hybrid sigma subunit directs RNA polymerase to a hybrid promoter in Escherichia coli. J Mol Biol 1995, 246:563-571. 30. Mencía M, Monsalve M, Rojo F, Salas M: Substitution of the C-terminal domain of the Escherichia coli RNA polymerase a subunit by that from Bacillus subtilis makes the enzyme responsive to a Bacillus subtilis transcriptional activator. J Mol Biol 1998, 275:177-185. 31. Fields S, Song O: A novel genetic system to detect protein–protein interactions. Nature 1989, 340:245-246. 32. Karimova G, Pidoux J, Ullman A, Ladant D: A bacterial two-hybrid •• system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci USA 1998, 95:5752-5756. This new system enables one to perform two-hybrid technology directly in E. coli. In addition, because the system relies on the generation of a regulatory molecule, the two interacting components need not be located in the vicinity of the DNA. 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Mesnage S, Tosi-Couture E, Fouet A: Production and cell surface • anchoring of functional fusions between the SLH motifs of the Bacillus anthracis S-layer proteins and the Bacillus subtilis levansucrase. Mol Microbiol 1999, 31:927-936. The first demonstration that SLH domains are sufficient to promote the attachment of foreign polypeptides on the surface of bacteria. 25. Murphy JR: Protein engineering and design for drug delivery. Curr Opin Struct Biol 1996, 6:541-545. 26. Coen L, Osta R, Maury M, Brulet P: Construction of hybrid proteins that migrate retrogradely and transynaptically into the central nervous system. Proc Natl Acad Sci USA 1997, 94:9400-9405. 43. Betton JM, Jacob JP, Hofnung M, Broome-Smith JK: Creating a bifunctional protein by insertion of a b-lactamase into maltodextrin-binding protein. Nat Biotechnol 1997, 15:1276-1279. 44. Brennan CA, Christianson K, La Fleur MA, Mandecki W: A molecular sensor system based on genetically engineered alkaline phosphatase. Proc Natl Acad Sci USA 1995, 92:5783-5787. 45. Fushinobu S, Ohta T, Matsuzawa H: Homotropic activation via the • subunit interaction and allosteric symmetry revealed on analysis of hybrid enzymes of L-lactate dehydrogenase. J Biol Chem 1998, 273:2971-2976. A recent paper demonstrating how the substrate specificity of a multimeric enzyme can be modified while retaining sensitivity to the original allosteric effector.