Tailoring New Enzyme Functions by Rational Redesign Frédéric Cedrone*, André Ménez*† and Eric Quéméneur‡

405

Tailoring new enzyme functions by rational redesign Frédéric Cedrone*, André Ménez*† and Eric Quéméneur‡

Site-directed mutagenesis is still a very efficient strategy to Figure 1 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 knowledge- based studies greatly benefit from the most recent computational analyses of enzyme structures and functions. Reinforcement of Change of The combination of rational and combinatorial methods opens a promiscuous substrate up new vistas in the design of stable and efficient enzymes. reaction specificity

Change of Addresses enzyme *CEA, Département d’Ingénierie et d’Etudes des Protéines, mechanism 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

Abbreviations BChE butyrylcholinesterase HSD hydroxysteroid dehydrogenase IGPS indole-3 glycerol phosphate synthase NTF2 nuclear transport factor 2 PDB Protein Data Bank Identification PRAI phosphoribosyl-anthranilate isomerase of a convergent mechanism Introduction Identification The rational design of new biocatalysts challenges our of an appropriate knowledge of protein biochemistry. If protein engineers template could demonstrate their ability to fashion an enzyme for any Refunctionalization of the template preconceived task, with a minimum number of trials and errors, and within a reasonable period of time, this would Current Opinion in Structural Biology provide evidence that our understanding of protein structure/function relationships has come of age. Are we already Schematic representation of the possible strategies for redesigning there? Yes, in general, if we restrict ourselves to the problem enzyme functions. The upper part of the figure illustrates the three possible routes to shift a pre-existing enzyme activity into an original of re-engineering enzyme specificity. The response must be one. The lower part illustrates an alternative strategy based on the more guarded, though, in terms of redesigning enzyme assembling of independent enzyme features, such as a substrate- mechanisms and we are certainly unable, as yet, to consider binding site and the catalytic machinery, to create a novel biocatalyst. the de novo design of catalysts for completely new reactions. The white symbols (cross, stars) depict functional amino acid residues Despite obvious difficulties, a number of remarkable suc- that must be either changed or grafted during the different processes. cesses 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 available. A rational approach may also work from a rea- review, which describes the most recent trends in the ratio- sonable model derived from sequence alignments [12]. We nal redesign of enzyme substrate specificity and should not forget, however, that it is not yet possible to re- mechanisms, as well as attempts to engineer biocatalysts engineer rationally the most comprehensively studied through molecular assembling. trypsin into a fully active chymotrypsin-like enzyme, despite an obvious evolutionary relationship [13]. Reshaping enzyme specificity Nevertheless, a number of recent examples nicely illus- By and large, reshaping a substrate-binding site [1–4] or trate current trends in specificity redesign. cofactor specificity [5–9], or positioning charged residues to favor one substrate relative to another one [10,11] is An original generic approach was proposed, combining achievable when a good three-dimensional structure is site-directed mutagenesis and chemical modification, to 406 Engineering and design

extend the specificity of subtilisin at site S1. A cysteine nitrile hydratase activity by approximately 104 relative to residue was introduced in this site to be modified by vari- that of the wild-type enzyme, although some amide hydrol- ous chemical groups. The nature of these groups controls ysis properties could still be measured [24]. A comparable substrate binding and, thus, enzyme specificity. The pref- substitution was performed in a mechanistically, but not erence for substrates with large hydrophobic amino acids structurally, related enzyme, asparagine synthetase B (such as phenylalanine) at P1 could be shifted to a prefer- (N74D). The benefit was lesser than in the case of papain, ence for small (such as alanine) or charged residues (such with a ratio of only 200 between the wild-type and the vari- as glutamic acid and arginine) by introducing branching ant [25]. Although the functional equivalence (convergent bulky or charged chemical groups, respectively [14•]. evolution) of residues Q19 and N74 could be postulated, these works showed the importance of their structural con- A linoleate 13-lipoxygenase was converted into a 9-lipoxy- text for the evolution of promiscuous mechanisms. genase by a single substitution, H608V, at the level of the residue that is supposed to be responsible for the positional 4-Chlorobenzoyl-CoA dehalogenase was rationally evolved specificity of the substrate [15•]. Bulky His608 was replaced to a crotonyl hydratase, an activity that is absent from the by a residue with a smaller sidechain, so that a basic residue wild-type enzyme [26••]. The mechanism of crotonase (probably R758) at the bottom of the active pocket becomes requires acid-base donors, thus two glutamate residues accessible. The carboxylate group of the lipid substrate, were introduced at positions 117 and 137 (Figure 2). which is normally outside the pocket, becomes able to inter- Unfavorable interactions between the introduced act with this basic residue, resulting in a head-to-tail sidechains and a five-residue segment inside the dehalo- orientation of the substrate. The catalytic iron atom, react- genase active site led to insolubility of the first set of ing by radical rearrangement at the same positional site, will variants. By substitution of the five amino acid segment thus lead to the different oxygenation site in the lipid. and the introduction of a proline before E137 to favor its positioning, a crotonase activity could be generated with The conversion of 3α-hydroxysteroid dehydrogenase an improvement higher than 64,000-fold. (3α-HSD) into 20α-hydroxysteroid dehydrogenase (20α-HSD) using loop chimaeras [16•] yielded a very effi- Work on butyrylcholinesterase (BChE) showed how an cient variant of 3α-HSD with the specificity of 20α-HSD enzyme can be transformed to take a potent inhibitor as a (androgens→progesterone). This result is all the more substrate [27]. Organophosphorous acid anhydride com- striking as individual mutations in the critical loops failed pounds are strong irreversible inhibitors of enzymes to yield the desired specificity, illustrating the need for the containing nucleophilic serine in their active site, particu- introduction of more complex determinants for the struc- larly acetylcholinesterase in the neuromuscular junction. ture-based prediction to be successful. Soman is the most potent of these inhibitors. The mechanism of BChE can be redirected to favor the hydrolysis of Finally, the simple substitution in dimethyl sulfoxide the phosphite triester inhibitor intermediate, as opposed to reductase of cysteine for the molybdenum-ligating serine its dealkylation and irreversible modification of BChE. residue almost completely shifted the enzyme to an adenosine N1-oxide reductase [17]. This illustrates the Enzyme engineering by molecular assembling large impact that small changes in enzyme structure have Another means to engineer enzyme functions may exist in on protein specificity. assembling the necessary components, that is, the catalytic machinery, a substrate-binding site and so on, on a Re-engineering catalytic mechanisms selected macromolecular template. Such examples can be The current view is that chemistry, not binding specificity, found in nature, mostly as fusions of independent mod- is the dominant factor in the evolution of new enzymatic ules, such as in hybrid systems [28], polyketide synthases activities [18,19•]. As a consequence, proteins with similar [29–31] or FokI-derived chimaeric restriction enzymes folds can support very different chemical reactions after the [32]. Though we will not expand on these systems here, it incorporation of new catalytic groups. This is the main route is noteworthy that peptides or small proteins can now be for protein engineers to orient an enzyme toward an alterna- redesigned for catalytic [33•,34] or binding functions [35]. tive mechanism. For example, a single mutation changes They might be exquisite building blocks for designing ∆4-3-ketosteroid-5β-reductase to 3α-HSD [20]; four substi- modular architectures in the future. tutions were enough to confer an oleate-hydroxylase activity on an oleate-desaturase and six substitutions sufficed to con- Inventory of robust catalytic machineries vert a hydroxylase to a desaturase [21]. Because many A prerequisite to convergent enzyme redesign is the iden- enzymes involve promiscuous mechanisms, the balance tification of the small number of catalytic devices that can between two reactions may be controlled to improve one work in various structural contexts. This narrow repertoire enzyme function compared with another one [22,23]. can be managed by computational analyses in order to offer valuable information to protein engineers. Thus, the The introduction of a catalytic residue, by the substitution TESS software searches through a dataset of PDB struc- Q19E, in papain increased the turnover number for its tures for user-defined combinations of atoms or Tailoring new enzyme functions by rational redesign Cedrone, Ménez and Quéméneur 407

Figure 2

Divergent re-engineering of 4-chlorobenzoyl- CoA dehalogenase into a crotonase-like (a) (b) O syn-hydratase [26••]. These two enzymes CoA-S belong to the same enoyl-CoA O hydratase/isomerase family; however, their CoA-S substrates, intermediates and products (shown in bold), as well as their mechanisms, are very different. (a) The mechanism of the Cl crotonase involves the concerted syn addition of a proton from Glu164 and a hydroxide from F64 water bound to Glu144 across the Si face of N the double bond of the substrate. (b) The E164 G114 H catalysis in 4-chlorobenzoyl-CoA A98 O N G117 dehalogenase is based on a multistep N E144 H O G141 reaction. Asp145 adds to the benzoyl ring to H O CoA-S N W137 form a Meisenhiemer intermediate that will H O H evolve to an arylated intermediate after O O departure of the chloride. Its subsequent CoA-S H O H hydrolysis will result from the attack of a water O molecule activated by His90. The rational Cl O D145 redesign of 4-chlorobenzoyl-CoA O dehalogenase was based on the import of two Crotonyl-CoA hydratase (crotonase) H H90 glutamic acid residues, mimicking E144 and H E164, and on the reshaping of the active site 4-Chlorobenzoyl-CoA dehalogenase (six residues) to improve positioning of the catalytic amino acids.

O O CoA-S CoA-S OH

OH Current Opinion in Structural Biology

residues [36]. The results have been compiled in the than bacterial P450 [43]. The resulting hybrid enzyme PROCAT database of three-dimensional active site coor- exhibits mammalian enzyme active site characteristics, dinates. Another computational search for consensus with the solubility property of the bacterial enzyme. This catalytic devices, such as the Ser–His–Asp catalytic triad, demonstrated the permissivity of this protein fold for mas- the His–His heme coordination site and the Cys2–His2 sive change. More strikingly perhaps, an efficient enzyme zinc finger pattern, revealed new examples of evolution- could be built from two independent chains, the bisection ary convergence [37]. site being inside the active site [44].

Searching for appropriate engineering templates Grafting catalytic machineries As previously mentioned, nature seems to recruit a limited In parallel to the classical transition-state analog approach number of protein folds for building a large variety of func- used to generate abzymes, it is possible to engineer cat- tions [38–42]. A recent review showed that some enzyme alytic antibodies by grafting functional residues into their superfamilies display remarkably divergent properties in paratopes. A protease that is able to cleave the small bac- terms of functions, while conserving some specific chemi- terial protein HPr was thus engineered from an cal properties. For example, proteins of the enolase family immunoglobulin single-chain variable fragment (scFv) share the ability to abstract the α proton of a carboxylate, after introducing three residues in the combining site: a whereas N-acetylneuraminate lyases utilize a ‘common lysine to increase the polarizability of the carbonyl group; electron sink’ and a Schiff base and so on [18]. a glutamate to increase the nucleophilicity of a nearby water molecule; and a histidine to provide a proton to the However, the possibilities offered by nature to protein leaving group [45]. The same group also demonstrated the engineers for the redesign of enzyme function seem to go possibility of engineering a ribonuclease from an anti- beyond this well-known subset of structures. For example, poly(rI) by the substitution of a residue positioned close to a soluble and functional chimaeric bacterial–human the 2′-hydroxyl of the ribose cycle into histidine [46]. In cytochrome P450 could be engineered to oxidize 4-chloro- both cases, however, the resulting activities are significant, toluene in 4-chlorobenzylalcohol with a higher activity though still modest. 408 Engineering and design

Figure 3

Divergent redesign of an IGPS scaffold in Deletion of Rational engineering PRAI by a combination of knowledge-based helix α0 of IGPS scaffold engineering and directed evolution •• to introduce the approaches [58 ]. The superimposition of the active site features IGPS and PRAI structures showed the of PRAI regions that have to be adjusted in the IGPS Replacement of loop scaffold to introduce PRAI’s isomerization β1α1 by shorter loops mechanism and to modify the putative, but (4–7 amino acids) incomplete, binding site for phosphoribosyl- harboring the common anthranilate. Thus, in addition to the deletion α GK motif of the 48 N-terminal residues forming helix 0 Exchange of loop β6α6 and the introduction of aspartic acid as a Asn184 → Asp β α with PRAI-like sequence general base in position 184, the 1 1 loop substitution GXGGXGQ was replaced by shorter variable sequences 'RATIONAL LIBRARY' (4–7 residues) including the common GK motif and the β6α6 connection was replaced In vitro evolution by the PRAI-like sequence GXGGXGQ. The Fine-tuning resulting ‘rational library’ was submitted to of PRAI activity 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 Positive screen in E. coli phosphoribosyl-anthranilate than the natural Genetic selection enzyme (k /K = 4.8 × 107 M–1s–1) and a of an active cat M complete loss of its native activity. engineered IGPS →‘ivePRAI’

Current Opinion in Structural Biology

Protein-bound metals are other robust catalytic devices. Another example of the robustness of the Ser–His–Asp Nearly one third of all known proteins are metalloproteins mechanism was illustrated by the convergent re-engineering and a sound knowledge of these is available for rational of Escherichia coli periplasmic cyclophilin, a peptidyl-prolyl experiments. The early software for modeling metal-bind- isomerase, into a proline endopeptidase [53]. ing sites in proteins was called Dezymer [47,48]. Dezymer proved successful in the design of an iron-binding site in The marriage of rational and combinatorial thioredoxin to generate a moderate superoxide dismutase redesign activity [49]. An interesting alternative is the construction Most enzymes elaborated by rational redesign do not reach of semisynthetic enzymes by a combination of site-direct- the expected catalytic performances because of our limited ed mutagenesis and chemical modification with mastery of protein folding, dynamics and stability. Indeed, metal-chelating groups, instead of by direct coordination subtle changes in the geometry of an active site suffice to by amino acid sidechains [50,51]. generate tremendous unpredicted consequences for enzyme function [54]. The fine-tuning of engineered The Benkovic group [52•] has explored the possibility of enzymes can only be fulfilled today by combinatorial creating a nonexistent activity in a structurally related approaches [55–57]. Even more powerful, perhaps, is the protein. Scytalone dehydratase and nuclear transport fac- combination of directed evolution with knowledge-based tor 2 (NTF2) share structural similarities, despite low engineering, as cogently illustrated recently by the re-engi- sequence homology, and have no common function. A neering of an efficient phosphoribosyl-anthranilate comparative structural analysis of the two proteins isomerase (PRAI) from the scaffold of indole-3 glycerol revealed that two key catalytic residues of scytalone phosphate synthase (IGPS) [58••]. Several points may dehydratase already exist in NTF2, namely H85 and explain this success. IGPS and PRAI possess the same D31. They represent the starting point for redesigning α/β-barrel template, with 22% sequence identity. This fold the rest of the NTF2 catalytic machinery. The efficien- offers the interesting feature that specificity and catalysis cy of the most active NTF2 variant was about could be addressed as independent entities. The two 10–6 µM–1 min–1 compared with 27 µM–1 min–1 for wild- enzymes catalyze two consecutive steps in the tryptophan type scytalone dehydratase. biosynthesis pathway and, thus, share a common ligand, the Tailoring new enzyme functions by rational redesign Cedrone, Ménez and Quéméneur 409

product of PRAI being the substrate of IGPS. Appropriate 11. Mouratou B, Kasper P, Gehring H, Christen P: Conversion of tyrosine phenol-lyase to dicarboxylic amino acid beta-lyase, an regions of IGPS have been tailored so that this enzyme can enzyme not found in Nature. J Biol Chem 1999, 274:1320-1325. receive the catalytic residues from PRAI and bind phos- 12. Cronin CN: Redesign of choline acetyltransferase specificity by phoribosyl-anthranilate (Figure 3). A low level of activity protein engineering. J Biol Chem 1998, 273:24465-24469. could already be detected at the stage of the rational library. 13. Perona JJ, Craik CS: Evolutionary divergence of substrate A subset of this library was subjected to DNA shuffling and specificity within the chymotrypsin-like serine protease fold. J Biol a ‘staggered extension procedure’, then sorted by genetic Chem 1997, 272:29987-29990. selection for PRAI complementation. The best variant 14. DeSantis G, Shang X, Jones JB: Toward tailoring the specificity of • the S1 pocket of subtilisin B. lentus: chemical modification of (ivePRAI) exhibits a sixfold higher catalytic efficiency with mutant enzymes as a strategy for removing specificity limitations. respect to phosphoribosyl-anthranilate than the natural Biochemistry 1999, 38:13391-13397. PRAI enzyme and completely looses its IGPS activity. The authors have generated a series of chemically modified mutants of subtilisin from B. lentus with enlarged specificities. The normal preference of subtilisin for large hydrophobic residues at P1 has been switched to a preference for small and charged amino acids by a combination of site-directed Conclusions mutagenesis and chemical modification at the level of site S1. The specifici- Impressive breakthroughs in enzyme redesign have been ty depends on the chemical properties of the grafted motif, which makes it rather versatile a priori. achieved by rational approaches in recent years. This suc- 15. Hornung E, Walther M, Kühn H, Feussner I: Conversion of cucumber cess reflects our burgeoning understanding of the • linoleate 13-lipoxygenase to a 9-lipoxygenating species by site- molecular basis of enzymatic functions. Certainly, the com- directed mutagenesis. Proc Natl Acad Sci USA 1999, 96:4192-4197. plete ab initio synthesis of custom-designed enzymes still The replacement of His608, thought to determine the positional specificity remains a challenge. Yet, the controlled use of powerful of cucumber linoleate 13-lipoxygenase, by a less space-filling residue altered combinatorial methods allied to knowledge-based strate- the lipoxygenation site and converted the enzyme to a linoleate 9-lipoxygenase. The H608V mutation may demask a positive charge at the bottom of gies is expected to open up new horizons in the the active site and allow a head-to-tail orientation of the fatty acid substrate. fascinating, but challenging field of enzyme engineering. The catalytic iron atom, reacting at the same positional site, leads to a different oxygenation site in the lipid. References and recommended reading 16. Ma H, Penning TM: Conversion of mammalian 3α-hydroxysteroid • dehydrogenase to 20α-hydroxysteroid dehydrogenase using loop Papers of particular interest, published within the annual period of review, chimeras: changing specificity from androgens to progestins. Proc have been highlighted as: Natl Acad Sci USA 1999, 96:11161-11166. • of special interest The substitution of residues forming the steroid-binding pocket in 3α-HSD •• of outstanding interest by those found in 20α-HSD failed to alter its specificity for androgens to progestins. 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