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Tailoring new enzyme functions by rational redesign Frédéric Cedrone*, André Ménez*† and Eric Quéméneur‡ Site-directed mutagenesis is still a very efficient strategy to elaborate improved enzymes. Recently, advances have been made in developing rational strategies aimed at reshaping enzyme specificities and mechanisms, and at engineering biocatalysts through molecular assembling. These knowledgebased studies greatly benefit from the most recent computational analyses of enzyme structures and functions. The combination of rational and combinatorial methods opens up new vistas in the design of stable and efficient enzymes.
Figure 1
Reinforcement of a promiscuous reaction
Change of substrate specificity Change of enzyme mechanism
Addresses *CEA, Département d’Ingénierie et d’Etudes des Protéines, Bâtiment 152, CE Saclay, F-91191 Gif-sur-Yvette, France † e-mail:
[email protected] ‡C EA, Département d'Ingénierie et d'Etudes des Protéines - SBTN, Bâtiment 170, CE Valrhô, F-30207 Bagnols-sur-Cèze, France; e-mail:
[email protected] Current Opinion in Structural Biology 2000, 10:405–410 0959-440X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations BChE butyrylcholinesterase HSD hydroxysteroid dehydrogenase IGPS indole-3 glycerol phosphate synthase NTF2 nuclear transport factor 2 PDB Protein Data Bank PRAI phosphoribosyl-anthranilate isomerase
Introduction The rational design of new biocatalysts challenges our knowledge of protein biochemistry. If protein engineers could demonstrate their ability to fashion an enzyme for any preconceived task, with a minimum number of trials and errors, and within a reasonable period of time, this would provide evidence that our understanding of protein structure/function relationships has come of age. Are we already there? Yes, in general, if we restrict ourselves to the problem of re-engineering enzyme specificity. The response must be more guarded, though, in terms of redesigning enzyme mechanisms and we are certainly unable, as yet, to consider the de novo design of catalysts for completely new reactions. Despite obvious difficulties, a number of remarkable successes have emerged in recent years, largely thanks to our ever-growing knowledge of protein structures and mechanisms. Figure 1 summarizes the operational outline of this review, which describes the most recent trends in the rational redesign of enzyme substrate specificity and mechanisms, as well as attempts to engineer biocatalysts through molecular assembling.
Reshaping enzyme specificity By and large, reshaping a substrate-binding site [1–4] or cofactor specificity [5–9], or positioning charged residues to favor one substrate relative to another one [10,11] is achievable when a good three-dimensional structure is
Identification of a convergent mechanism Identification of an appropriate template Refunctionalization of the template Current Opinion in Structural Biology
Schematic representation of the possible strategies for redesigning enzyme functions. The upper part of the figure illustrates the three possible routes to shift a pre-existing enzyme activity into an original one. The lower part illustrates an alternative strategy based on the assembling of independent enzyme features, such as a substratebinding site and the catalytic machinery, to create a novel biocatalyst. The white symbols (cross, stars) depict functional amino acid residues that must be either changed or grafted during the different processes.
available. A rational approach may also work from a reasonable model derived from sequence alignments [12]. We should not forget, however, that it is not yet possible to reengineer rationally the most comprehensively studied trypsin into a fully active chymotrypsin-like enzyme, despite an obvious evolutionary relationship [13]. Nevertheless, a number of recent examples nicely illustrate current trends in specificity redesign. An original generic approach was proposed, combining site-directed mutagenesis and chemical modification, to
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extend the specificity of subtilisin at site S1. A cysteine residue was introduced in this site to be modified by various chemical groups. The nature of these groups controls substrate binding and, thus, enzyme specificity. The preference for substrates with large hydrophobic amino acids (such as phenylalanine) at P1 could be shifted to a preference for small (such as alanine) or charged residues (such as glutamic acid and arginine) by introducing branching bulky or charged chemical groups, respectively [14•]. A linoleate 13-lipoxygenase was converted into a 9-lipoxygenase by a single substitution, H608V, at the level of the residue that is supposed to be responsible for the positional specificity of the substrate [15•]. Bulky His608 was replaced by a residue with a smaller sidechain, so that a basic residue (probably R758) at the bottom of the active pocket becomes accessible. The carboxylate group of the lipid substrate, which is normally outside the pocket, becomes able to interact with this basic residue, resulting in a head-to-tail orientation of the substrate. The catalytic iron atom, reacting by radical rearrangement at the same positional site, will thus lead to the different oxygenation site in the lipid. The conversion of 3α-hydroxysteroid dehydrogenase (3α-HSD) into 20α-hydroxysteroid dehydrogenase (20α-HSD) using loop chimaeras [16•] yielded a very efficient variant of 3α-HSD with the specificity of 20α-HSD (androgens→progesterone). This result is all the more striking as individual mutations in the critical loops failed to yield the desired specificity, illustrating the need for the introduction of more complex determinants for the structure-based prediction to be successful. Finally, the simple substitution in dimethyl sulfoxide reductase of cysteine for the molybdenum-ligating serine residue almost completely shifted the enzyme to an adenosine N1-oxide reductase [17]. This illustrates the large impact that small changes in enzyme structure have on protein specificity.
Re-engineering catalytic mechanisms The current view is that chemistry, not binding specificity, is the dominant factor in the evolution of new enzymatic activities [18,19•]. As a consequence, proteins with similar folds can support very different chemical reactions after the incorporation of new catalytic groups. This is the main route for protein engineers to orient an enzyme toward an alternative mechanism. For example, a single mutation changes ∆4-3-ketosteroid-5β-reductase to 3α-HSD [20]; four substitutions were enough to confer an oleate-hydroxylase activity on an oleate-desaturase and six substitutions sufficed to convert a hydroxylase to a desaturase [21]. Because many enzymes involve promiscuous mechanisms, the balance between two reactions may be controlled to improve one enzyme function compared with another one [22,23]. The introduction of a catalytic residue, by the substitution Q19E, in papain increased the turnover number for its
nitrile hydratase activity by approximately 104 relative to that of the wild-type enzyme, although some amide hydrolysis properties could still be measured [24]. A comparable substitution was performed in a mechanistically, but not structurally, related enzyme, asparagine synthetase B (N74D). The benefit was lesser than in the case of papain, with a ratio of only 200 between the wild-type and the variant [25]. Although the functional equivalence (convergent evolution) of residues Q19 and N74 could be postulated, these works showed the importance of their structural context for the evolution of promiscuous mechanisms. 4-Chlorobenzoyl-CoA dehalogenase was rationally evolved to a crotonyl hydratase, an activity that is absent from the wild-type enzyme [26••]. The mechanism of crotonase requires acid-base donors, thus two glutamate residues were introduced at positions 117 and 137 (Figure 2). Unfavorable interactions between the introduced sidechains and a five-residue segment inside the dehalogenase active site led to insolubility of the first set of variants. By substitution of the five amino acid segment and the introduction of a proline before E137 to favor its positioning, a crotonase activity could be generated with an improvement higher than 64,000-fold. Work on butyrylcholinesterase (BChE) showed how an enzyme can be transformed to take a potent inhibitor as a substrate [27]. Organophosphorous acid anhydride compounds are strong irreversible inhibitors of enzymes containing nucleophilic serine in their active site, particularly acetylcholinesterase in the neuromuscular junction. Soman is the most potent of these inhibitors. The mechanism of BChE can be redirected to favor the hydrolysis of the phosphite triester inhibitor intermediate, as opposed to its dealkylation and irreversible modification of BChE.
Enzyme engineering by molecular assembling Another means to engineer enzyme functions may exist in assembling the necessary components, that is, the catalytic machinery, a substrate-binding site and so on, on a selected macromolecular template. Such examples can be found in nature, mostly as fusions of independent modules, such as in hybrid systems [28], polyketide synthases [29–31] or FokI-derived chimaeric restriction enzymes [32]. Though we will not expand on these systems here, it is noteworthy that peptides or small proteins can now be redesigned for catalytic [33•,34] or binding functions [35]. They might be exquisite building blocks for designing modular architectures in the future. Inventory of robust catalytic machineries
A prerequisite to convergent enzyme redesign is the identification of the small number of catalytic devices that can work in various structural contexts. This narrow repertoire can be managed by computational analyses in order to offer valuable information to protein engineers. Thus, the TESS software searches through a dataset of PDB structures for user-defined combinations of atoms or
Tailoring new enzyme functions by rational redesign Cedrone, Ménez and Quéméneur
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Figure 2 Divergent re-engineering of 4-chlorobenzoylCoA dehalogenase into a crotonase-like syn-hydratase [26••]. These two enzymes belong to the same enoyl-CoA hydratase/isomerase family; however, their substrates, intermediates and products (shown in bold), as well as their mechanisms, are very different. (a) The mechanism of the crotonase involves the concerted syn addition of a proton from Glu164 and a hydroxide from water bound to Glu144 across the Si face of the double bond of the substrate. (b) The catalysis in 4-chlorobenzoyl-CoA dehalogenase is based on a multistep reaction. Asp145 adds to the benzoyl ring to form a Meisenhiemer intermediate that will evolve to an arylated intermediate after departure of the chloride. Its subsequent hydrolysis will result from the attack of a water molecule activated by His90. The rational redesign of 4-chlorobenzoyl-CoA dehalogenase was based on the import of two glutamic acid residues, mimicking E144 and E164, and on the reshaping of the active site (six residues) to improve positioning of the catalytic amino acids.
(a)
(b)
O CoA-S
O CoA-S Cl F64 E164 A98 O N G141 H O N O H H O O H O H CoA-S
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G114 N H CoA-S
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Crotonyl-CoA hydratase (crotonase)
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residues [36]. The results have been compiled in the PROCAT database of three-dimensional active site coordinates. Another computational search for consensus catalytic devices, such as the Ser–His–Asp catalytic triad, the His–His heme coordination site and the Cys2–His2 zinc finger pattern, revealed new examples of evolutionary convergence [37].
than bacterial P450 [43]. The resulting hybrid enzyme exhibits mammalian enzyme active site characteristics, with the solubility property of the bacterial enzyme. This demonstrated the permissivity of this protein fold for massive change. More strikingly perhaps, an efficient enzyme could be built from two independent chains, the bisection site being inside the active site [44].
Searching for appropriate engineering templates
Grafting catalytic machineries
As previously mentioned, nature seems to recruit a limited number of protein folds for building a large variety of functions [38–42]. A recent review showed that some enzyme superfamilies display remarkably divergent properties in terms of functions, while conserving some specific chemical properties. For example, proteins of the enolase family share the ability to abstract the α proton of a carboxylate, whereas N-acetylneuraminate lyases utilize a ‘common electron sink’ and a Schiff base and so on [18].
In parallel to the classical transition-state analog approach used to generate abzymes, it is possible to engineer catalytic antibodies by grafting functional residues into their paratopes. A protease that is able to cleave the small bacterial protein HPr was thus engineered from an immunoglobulin single-chain variable fragment (scFv) after introducing three residues in the combining site: a lysine to increase the polarizability of the carbonyl group; a glutamate to increase the nucleophilicity of a nearby water molecule; and a histidine to provide a proton to the leaving group [45]. The same group also demonstrated the possibility of engineering a ribonuclease from an antipoly(rI) by the substitution of a residue positioned close to the 2′-hydroxyl of the ribose cycle into histidine [46]. In both cases, however, the resulting activities are significant, though still modest.
However, the possibilities offered by nature to protein engineers for the redesign of enzyme function seem to go beyond this well-known subset of structures. For example, a soluble and functional chimaeric bacterial–human cytochrome P450 could be engineered to oxidize 4-chlorotoluene in 4-chlorobenzylalcohol with a higher activity
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Figure 3
Rational engineering of IGPS scaffold to introduce the active site features of PRAI
Deletion of helix α0
Replacement of loop β1α1 by shorter loops (4–7 amino acids) harboring the common GK motif
Exchange of loop β6α6 Asn184 → Asp with PRAI-like sequence substitution GXGGXGQ 'RATIONAL LIBRARY' In vitro evolution
Fine-tuning of PRAI activity
Positive screen in E. coli
Genetic selection of an active engineered IGPS →‘ivePRAI’
Divergent redesign of an IGPS scaffold in PRAI by a combination of knowledge-based engineering and directed evolution approaches [58••]. The superimposition of the IGPS and PRAI structures showed the regions that have to be adjusted in the IGPS scaffold to introduce PRAI’s isomerization mechanism and to modify the putative, but incomplete, binding site for phosphoribosylanthranilate. Thus, in addition to the deletion of the 48 N-terminal residues forming helix α0 and the introduction of aspartic acid as a general base in position 184, the β1α1 loop was replaced by shorter variable sequences (4–7 residues) including the common GK motif and the β6α6 connection was replaced by the PRAI-like sequence GXGGXGQ. The resulting ‘rational library’ was submitted to genetic selection by complementing an E. coli strain lacking PRAI activity and to in vitro evolution, which generated eight substitutions (★) and one deletion (✩). Finally, the selected IGPS variant (ivePRAI) exhibits a sixfold higher catalytic efficiency with respect to phosphoribosyl-anthranilate than the natural enzyme (kcat/KM = 4.8 × 107 M–1s–1) and a complete loss of its native activity.
Current Opinion in Structural Biology
Protein-bound metals are other robust catalytic devices. Nearly one third of all known proteins are metalloproteins and a sound knowledge of these is available for rational experiments. The early software for modeling metal-binding sites in proteins was called Dezymer [47,48]. Dezymer proved successful in the design of an iron-binding site in thioredoxin to generate a moderate superoxide dismutase activity [49]. An interesting alternative is the construction of semisynthetic enzymes by a combination of site-directed mutagenesis and chemical modification with metal-chelating groups, instead of by direct coordination by amino acid sidechains [50,51]. The Benkovic group [52•] has explored the possibility of creating a nonexistent activity in a structurally related protein. Scytalone dehydratase and nuclear transport factor 2 (NTF2) share structural similarities, despite low sequence homology, and have no common function. A comparative structural analysis of the two proteins revealed that two key catalytic residues of scytalone dehydratase already exist in NTF2, namely H85 and D31. They represent the starting point for redesigning the rest of the NTF2 catalytic machinery. The efficiency of the most active NTF2 variant was about 10–6 µM–1 min–1 compared with 27 µM–1 min–1 for wildtype scytalone dehydratase.
Another example of the robustness of the Ser–His–Asp mechanism was illustrated by the convergent re-engineering of Escherichia coli periplasmic cyclophilin, a peptidyl-prolyl isomerase, into a proline endopeptidase [53].
The marriage of rational and combinatorial redesign Most enzymes elaborated by rational redesign do not reach the expected catalytic performances because of our limited mastery of protein folding, dynamics and stability. Indeed, subtle changes in the geometry of an active site suffice to generate tremendous unpredicted consequences for enzyme function [54]. The fine-tuning of engineered enzymes can only be fulfilled today by combinatorial approaches [55–57]. Even more powerful, perhaps, is the combination of directed evolution with knowledge-based engineering, as cogently illustrated recently by the re-engineering of an efficient phosphoribosyl-anthranilate isomerase (PRAI) from the scaffold of indole-3 glycerol phosphate synthase (IGPS) [58••]. Several points may explain this success. IGPS and PRAI possess the same α/β-barrel template, with 22% sequence identity. This fold offers the interesting feature that specificity and catalysis could be addressed as independent entities. The two enzymes catalyze two consecutive steps in the tryptophan biosynthesis pathway and, thus, share a common ligand, the
Tailoring new enzyme functions by rational redesign Cedrone, Ménez and Quéméneur
product of PRAI being the substrate of IGPS. Appropriate regions of IGPS have been tailored so that this enzyme can receive the catalytic residues from PRAI and bind phosphoribosyl-anthranilate (Figure 3). A low level of activity could already be detected at the stage of the rational library. A subset of this library was subjected to DNA shuffling and a ‘staggered extension procedure’, then sorted by genetic selection for PRAI complementation. The best variant (ivePRAI) exhibits a sixfold higher catalytic efficiency with respect to phosphoribosyl-anthranilate than the natural PRAI enzyme and completely looses its IGPS activity.
Conclusions Impressive breakthroughs in enzyme redesign have been achieved by rational approaches in recent years. This success reflects our burgeoning understanding of the molecular basis of enzymatic functions. Certainly, the complete ab initio synthesis of custom-designed enzymes still remains a challenge. Yet, the controlled use of powerful combinatorial methods allied to knowledge-based strategies is expected to open up new horizons in the fascinating, but challenging field of enzyme engineering.
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