8-Barrel Enzymes John a Gerlt� and Frank M Raushely

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Evolution of function in (b/a)8-barrel enzymes John A Gerlt and Frank M Raushely

The (b/a)8-barrel is the most common fold in structurally closed, parallel b-sheet structure of the (b/a)8-barrel is characterized enzymes. Whether the functionally diverse formed from eight parallel (b/a)-units linked by hydrogen enzymes that share this fold are the products of either divergent bonds that form a cylindrical core. Despite its eightfold or convergent evolution (or both) is an unresolved question that pseudosymmetry, the packing within the interior of the will probably be answered as the sequence databases continue barrel is better described as four (b/a)2-subdomains in to expand. Recent work has examined natural, designed, and which distinct hydrophobic cores are located between the directed evolution of function in several superfamilies of (b/a)8- b-sheets and flanking a-helices [2]. barrel containing enzymes. The active sites are located at the C-terminal ends of the Addresses b-strands. So placed, the functional groups surround the Departments of Biochemistry and Chemistry, University of Illinois at active site and are structurally independent: the ‘old’ and Urbana-Champaign, 600 South Mathews Avenue, Urbana, ‘new’ enzymes retain functional groups at the ends of Illinois 61801, USA e-mail: [email protected] some b-strands, but others are altered to allow the ‘new’ yDepartment of Chemistry, Texas A&M University, PO Box 30012, activity [3]. With this blueprint, the (b/a)8-barrel is opti- College Station, Texas 77842-3012, USA mized for evolution of new functions. With a rapidly e-mail: [email protected] increasing number of sequences and structures, the challenge is to understand how nature uses the (b/a)8-barrel scaffold in natural evolution so that in vitro methods can Current Opinion in Chemical Biology 2003, 7:252–264 be used to devise catalysts for new reactions. This review comes from a themed section on Biocatalysis and biotransformation Using both structural inspection and substrate specificity, Edited by Tadhg Begley and Ming-Daw Tsai the SCOP database (http://scop.mrc-lmb.cam.ac.uk/scop/) 1367-5931/03/$ – see front matter clusters proteins into ‘superfamilies’ that evolved from a ß 2003 Elsevier Science Ltd. All rights reserved. common ancestor; the clustering assumes that divergent evolution retains discernible structural similarity even if DOI 10.1016/S1367-5931(03)00019-X sequence similarity has disappeared [4]. In the most recent release (1.61; November 2002), (b/a)8-barrel pro- Abbreviations teins were clustered into 25 superfamilies. Given that the BLAST basic local alignment sequence tool DHO dihydroorotase topologies of all (b/a)8-barrels are similar (closed, cylind- HisA phosphoribosylformimino-5-aminoimidazole carboxamide rical structures) and that retention of substrate specificity ribonucleotide isomerase need not be important in evolutionary processes, the HisF imidazole glycerolphosphate synthase SCOP database risks either placing homologous enzymes GalD D-galactonate dehydratase into separate superfamilies or placing non-homologous GlucD D-glucarate dehydratase HPS D-arabino-hex-3-ulose 6-phosphate synthase enzymes into the same superfamily. HYD hydantoinase KDGP 2-keto-3-deoxy-6-phosphogluconate Using sequence relationships detected with PSI-BLAST, KGPDC 3-keto-L-gulonate 6-phosphate decarboxylase Copley and Bork [5] concluded that 12 of the super- MAL 3-methylaspartate ammonia lyase families are derived from a common ancestor. Are the MLE muconate lactonizing enzyme MR mandelate racemase remaining superfamilies the result of convergent evolu- 0 OMP orotidine 5 -monophosphate tion? Or have all (b/a)8-barrel enzymes diverged from a OSBS o-succinylbenzoate synthase common ancestor? These questions can be answered 0 PRAI N-(5 -phosphoribosyl)anthranilate isomerase as sequences and structures of ‘new’ proteins provide PSI-BLAST position sensitive interactive BLAST PTE phosphotriesterase ‘missing links’ to connect the superfamilies. RhamD L-rhamnonate dehydratase RPE D-ribulose 5-phophate 3-epimerase Evolution from libraries of fractional SCOP Structural Classification of Proteins barrels? URE urease The (b/a)2-subdomain structure allows the suggestion that (b/a)8-barrels are assembled from (b/a)2N-precursors. Introduction Mixing precursors that separately deliver binding or cat- The (b/a)8-barrel fold is the most common in enzymes, alysis might produce the progenitors of superfamilies. with 10% of structurally characterized proteins contain- After that process, evolution of function could occur by ing at least one domain with this fold (Figure 1) [1]. The gene duplication followed by mutation.

Current Opinion in Chemical Biology 2003, 7:252–264 www.current-opinion.com Evolution of function in (b/a)8-barrel enzymes Gerlt and Raushel 253

Figure 1

The (b/a)8-barrel in yeast triosephosphate isomerase (7TIM.pdb) viewed from (a) the C-terminal ends of the b-strands and (b) from the side.

Evidence to support the formation of (b/a)8-barrels from How is the progenitor chosen? Two criteria can be envi- (b/a)2N-subdomains includes: saged: 1. HisA and HisF, which catalyse successive reactions in 1. The progenitor ‘accidentally’ catalyzes the desired histidine biosynthesis, are homologous in both se- new reaction, probably at a low but sufficient rate to quence and structure [6]. Each were formed by fusion provide selective advantage, immediately after gene of two equivalent (b/a)4-units. The N- and C-terminal duplication. Such catalytic promiscuity apparently (b/a)4-units in HisF are stable polypeptides that form is wide-spread (e.g. a protein that catalyzes the o- both homodimeric and heterodimeric structures in succinylbenzoate synthase (OSBS) reaction in mena- solution [7]. quinone biosynthesis was first identified by its 2. With few exceptions, barrels have the (b/a)8-fold. The N-acylamino acid racemase activity [11]). pertinent exceptions are enolase with a baab(b/a)6- 2. The progenitor does not catalyse the desired new barel fold [8] and quinolinate phosphoribosyltransfer- reaction, but a limited number of mutations (one?) ase with a (b/a)6-fold [9]. These irregular barrels can be yields the ‘new’ function, presumably at the expense explained by modular construction that deviates in of the original reaction. When this mutation occurs, detail from the usual (b/a)8-barrel. selection is possible and optimization can proceed. 3. A search of the Protein Data Bank with the (b/a)4-half Until recently, no example of such evolutionary poten- barrels of HisA and HisF revealed sequence and struc- tial had been discovered in enzymes that possess the ture similarity with a (b/a)5-flavodoxin-like domain (b/a)8-barrel fold [12 ]. found in several proteins [10]. The discovery of this Three strategies can be envisaged for the relationship homologous substructure supports the hypothesis that a between the reactions catalyzed by the progenitor and progenitor (b/a) -structure existed. 4 ‘new’ enzyme [13]: Irrespective of whether the formation of (b/a)8-barrels occurs in toto or in pieces, the strategies that nature uses in 1. Chemical mechanism is dominant. The mechanism divergent evolution of intact (b/a)8-barrels have impor- of progenitor can be used to catalyse the ‘new’ reac- tant implications for genome annotation and de novo tion. In the simplest example, the progenitor and design of catalysts. ‘new’ enzyme catalyse the same chemical reaction but differ in substrate specificity. In more complex Natural evolution examples, the reactions share a structural strategy to A reasonable pathway for evolution of function involves stabilize a common intermediate or transition state. duplication of the gene encoding the progenitor so that The ‘enolase C-terminal domain-like’ and ‘metallo- the original function can be retained while the duplicate dependent hydrolases’ superfamilies provide exam- undergoes divergence of sequence and function. ples of this strategy. www.current-opinion.com Current Opinion in Chemical Biology 2003, 7:252–264 254 Biocatalysis and biotransformation

2. Ligand specificity is dominant. Metabolic pathways Recently, homologues of MLE encoded by the Escherichia can evolve ‘linearly’ (from start to finish or vice versa)so coli and Bacillus subtilis genomes were assigned a novel the progenitor and ‘new’ enzymes share a substrate/ function. Although MLE catalyzes cycloisomerization and product. Enzymes in histidine and tryptophan bio- the homologous OSBS catalyzes dehydration, the OSBS synthesis are examples of this strategy. The SCOP from Amycolaptosis also catalyzes racemization of N-acyl- database assumes such a strategy in the ‘ribulose- amino acids [11]. Genome context as well as the 1,1-proton phosphate binding’ and ‘phosphoenol pyruvate/pyru- transfer reaction catalyzed by the promiscuous OSBS were vate domain’ superfamilies. used to deduce the L-Ala-D/L-Glu epimerase function 3. Active site architecture is dominant. Nature selects a [15,16]. L-Ala-D-Glu is a constituent of the murein peptide, progenitor with functional groups that can be used to and the epimerase, endopeptidases, and binding proteins catalyse a mechanistically distinct ‘new’ reaction. The encoded by proximal genes apparently are involved in first example of this opportunistic strategy is provided utilization of the murein peptide as carbon source. by orotidine 50-monophosphate (OMP) decarboxylase and 3-keto-L-gulonate 6-phosphate decarboxylase The ‘canonical’ acid/base catalysts found in enolase, (KGPDC), mechanistically distinct decarboxylases MLE, and MR are not sufficient to catalyse the range that have homologous sequences and structures. of reactions associated with the enolase superfamily. D-Galactonate dehydratase (GalD) from E. coli catalyzes anti-dehydration involving base-catalyzed proton abstrac- Enolase superfamily tion from C-2 and acid-catalyzed departure of the hydro- The ‘enolase C-terminal domain-like’ superfamily in xide leaving group from C-3. GalD contains the conserved SCOP contains those enzymes that had been recognized His–Asp general base but is lacking the acidic lysine at the as homologues using strict structural and mechanistic end of the second b-strand. Instead, His185, a ‘new’ criteria. This superfamily is the paradigm for superfami- general acid, and Asp183, a Mg2þ ligand, are located at lies whose members catalyse different reactions sharing a the end of the third b-strand [17]. common partial reaction: general-base catalyzed formation of an enolic intermediate that is stabilized by coor- A syn-dehydration is catalyzed by D-glucarate dehydratase dination to an active site Mg2þ; depending on the (GlucD) from E. coli; a single acid/base, the His339– identity of the reaction, the intermediate is partitioned Asp313 dyad, has been implicated as the base for proton to different products using either conserved or reaction- abstraction as well as the acid for hydroxide departure specific catalysts [14]. The members share two conserved [18,19]. L-Rhamnonate dehydratase (RhamD) from E. coli domains: an N-terminal domain that determines sub- also catalyzes syn-dehydration. In this case, the available strate specificity; and a (b/a)8-barrel domain that contains data point to base-catalyzed proton abstraction from C-2 the catalytic groups. Conservation of the bidomain struc- by the conserved His329–Asp302 dyad. However, the ture provides compelling evidence that the enolase ‘new’ acid is probably His281, located at the end of superfamily represents divergent evolution from a com- the fifth b-strand (following the Asp280 ligand for the mon progenitor. essential Mg2þ). Why two enzymes that catalyse syn- dehydration use different acid catalysts is unknown; The members are partitioned into three groups on the however, this mystery underscores the difficulty in de- basis of the identities of the three metal ion ligands ducing function from sequence or structure alone. (located at the ends of the third, fourth, and fifth b- strands) and the acid/base catalysts. The enolases contain 3-Methylaspartate ammonia lyase (MAL), which cata- a lysine (at the end of the sixth b-strand) that catalyzes lyzes anti-deamination, was proposed to be a member formation of the enolic intermediate; the general acid is of the enolase superfamily on the basis of limited located on a long loop inserted after the second b-strand. sequence homology: only four residues, the ligands for Other members are similar to muconate lactonizing the essential Mg2þ and the lysine general base, are enzyme (MLE) and contain lysine acid/base catalysts conserved in MAL and other members [14]. Structural on opposite sides of the active site (at the ends of the studies of two MALs revealed that these share the bido- second and sixth b-strands). Others are most similar to main structure characteristic of the enolase superfamily, mandelate racemase (MR) and contain a His–Asp dyad (at confirming their membership in the superfamily [20,21]. the ends of the seventh and sixth b-strands, respectively) MAL, like enolase, which catalyzes anti-dehydration, and often, but not always, a lysine on the opposite side of utilizes a lysine at the end of the sixth b-strand as the the active site (at the end of the second b-strand). base, but no acid group is located on the opposite face of the active site; the ammonia leaving group does not An understanding of the range of functions catalyzed require protonation for departure. by members of the enolase superfamily is essential for dissecting the structural changes involved in divergent Eleven reactions now have been associated with the evolution. enolase superfamily: enolase; MR and five acid sugar

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dehydratases (GalD, GlucD, RhamD, D-altronate dehy- has not been identified thus far. In at least one enzyme, dratase, D-gluconate dehydratase; C Millikin and JA Gerlt, designated as the PTE homology protein (PHP), the unpublished results) that constitute the MR subgroup; bridging lysine residue has been replaced by a glutamate MLE, OSBS, and L-Ala-D/L-Glu epimerase that constitute residue as illustrated in Figure 4b [25]. Although the the MLE subgroup; and MAL (Figure 2). The databases function of this protein is unknown, this structural per- contain > 380 members of the superfamily, excluding the turbation suggests that a carbamate functional group is ubiquitous enolases. In some cases, ‘old’ (orthologous) not required to bridge the two metal ions. functions can be assigned from sequence identity and genome context. However, the identities of most are With adenosine and cytosine deaminase [26], the brid- unknown, and these often contain active site motifs that ging ligand is missing altogether and the protein appar- differ from those established for the ‘old’ functions. The ently functions with a single divalent cation in the active challenge is assignment of ‘new’ functions that expand our site. This perturbation illustrates the structural and func- understanding of the limits of natural evolution. tional plasticity of the active site within the amidohydrolase superfamily of enzymes. The other active site Amidohydrolase superfamily ligands (four histidines and the aspartic acid) are present The ‘metallo-dependent hydrolase’ superfamily in SCOP in the subfamily but the catalytic functions have been was designated as the ‘amidohydrolase superfamily’ by altered. The structure of the mononuclear metal centre is Holm and Sander [22]. Subsequent analyses have illustrated in Figure 4c for cytosine deaminase [26]. Here revealed that members of this superfamily catalyse the the single divalent cation is ligated to two histidine hydrolysis of a wide variety of substrates at tetrahedral residues from the first b-strand and an aspartic acid from phosphorus and trigonal carbon centres. A partial list of the eighth b-strand. However, the histidine from the fifth substrates recognized by members of this superfamily of b-strand now serves as the final ligand to the lone metal enzymes is graphically depicted in Figure 3. ion. The histidine from the fourth b-strand does not ligate to the metal ion but functions as a general base catalyst for The hallmark for this superfamily of enzymes is a mono- the activation of the nucleophilic water molecule. nuclear or binuclear metal centre that functions to activate a hydrolytic water molecule for nucleophilic attack. In The most structurally diverse example identified to date some instances the metal centre also serves to enhance is found within the active site of human renal dipeptidase the electrophilic character of the substrate. In all members [27]. This enzyme binds two divalent cations but the of this superfamily the metal centre is found at the C- bridging lysine residue has been replaced by a glutamic terminal end of the central (b/a)8-barrel domain. The most acid. Moreover, one of the histidine residues from the first common structural motif identified thus far for enzymes b-strand and the aspartic acid from the eighth b-strand with two divalent cations is the one represented by have been replaced by a single aspartic acid from the first dihydroorotase (DHO), as illustrated in Figure 4a [23]. b-strand. The structure of this binuclear metal centre is Virtually identical binuclear metal centres are found in presented in Figure 4d. phosphotriesterase (PTE), urease (URE) and hydantoinase (HYD). In this particular example, the two metal The identity of the specific metal ion activators within the ions in DHO are separated by about 3.5 A˚ . The more amidohydrolase superfamily has not been conserved. 2þ solvent-shielded metal ion (Ma) is ligated to two histidine With URE there appears to be a requirement for Ni residues from the first b-strand and an aspartic acid from [28], whereas with PTE, DHO, and HYD the ‘natural’ the eighth b-strand. The more solvent-exposed metal ion metal ion activator appears to be Zn2þ, although PTE 2þ 2þ 2þ 2þ (Mb) is ligated to two histidine residues from the fifth and functions perfectly well with Co ,Ni ,Cd or Mn sixth b-strands. In addition to these five ligands, the metal [29]. Cytosine deaminase requires Fe2þ for activity, ions are bridged by a carbamate functional group formed although the structurally and functional equivalent ade- from the post-translational carboxylation of the e-amino nosine deaminase requires Zn2þ [26]. group of a lysine from the fourth b-strand. Finally the two metal ions are bridged by a hydroxide from solvent. What is not so clear is the functional requirement for either a binuclear or mononuclear metal centre. The conservation The functional significance of the carboxylated lysine of the metal ligands for these two subfamilies clearly residue that bridges the two metal ions is not clear. This indicates a divergence from a common ancestral parent. particular post-translational modification might serve as a One plausible functional requirement may emanate from control mechanism for the regulation of catalytic activity the reactions catalyzed by these two systems. With ade- or it might simply be an evolutionary relic. In vitro nosine and cytosine deaminases, cleavage of the C–N experiments with PTE have demonstrated that the bond requires the protonation of both products and thus binuclear metal centre will self-assemble in the presence the resting state of the protein may require a bound water of bicarbonate and the appropriate divalent cation [24]. for the supply of both protons. By contrast, with DHO and However, a protein catalyst for the carboxylation reaction URE, only the amino group product requires protonation www.current-opinion.com Current Opinion in Chemical Biology 2003, 7:252–264 256 Biocatalysis and biotransformation

Figure 2

2− 2− − − OPO3 OPO3 CO2 MR CO2 − Enolase O C −O C 2 2 OH H CH OH −H O 2 2 CH OH H anti 2 H 2-PGA PEP (R)-Mandelate (S)-Mandelate − − CO2 CO2 HO H O − HO H GlucD H H O H H H H MLE H O H OH − H OH O C O C H2O HO H HO H − − syn CO2 CO2 − − CO2 CO2

cis,cis-Muconate Muconolactone D-Glucarate

− CO2 O HO H H − − H OH O CO2 CO OSBS 2 H HO H GlucD H H − − CO − CO 2 H2O 2 H OH − H OH H2O O O HO H anti HO H − − CO2 CO2 SHCHC OSB L-Idarate

− − − − CO2 CO2 O2C O2C AE Epim H OH O − − O CO2 CO2 HO H GalD H H N O H HO H − H O HO H Ala H H NH 2 H OH anti H OH Ala CH2OH CH2OH L-Ala-L-Glu L-Ala-D-Glu D-Galactonate − − CO2 CO2 − H OH O CO2 − RhamD O C CH H OH H H H CH3 MAL 2 3 − HO H + HO H H2O H3N H − NH3 − HO H syn HO H − H CO2 CO2 anti CH3 CH3

3-Methyl Asp Mesaconate L-Rhamnonate − − CO2 CO2 HO H O H OH AltD H H H OH − H OH H2O H OH anti H OH

CH2OH CH2OH

D-Altronate

− − CO2 CO2 H OH O HO H GlcD H H − H OH H OH H2O H OH anti H OH

CH2OH CH2OH

D-Gluconate Current Opinion in Chemical Biology

Current Opinion in Chemical Biology 2003, 7:252–264 www.current-opinion.com Evolution of function in (b/a)8-barrel enzymes Gerlt and Raushel 257

Figure 3 members of the ‘ribulose-phosphate binding barrel’ superfamily as well as ‘missing links’ that provide per- O suasive evidence that OMP decarboxylase and RPE are O O homologous enzymes. OMP decarboxylases are homolo- EtO P OH NO HN 2 gous to both D-arabino-hex-3-ulose 6-phosphate synthase H2N NH2 OEt ON COOH (HPS) and KGPDC, the latter incorrectly annotated as Urease Phosphotriesterase H ‘probable hexulose phosphate synthases’ in several bac- Dihydroorotase terial genomes. These constitute the members of the Cl NH OMP decarboxylase suprafamily, the members of which RO 2 N catalyse different overall reactions with unrelated N N N HN NH mechanisms (Figure 5) [12,13]. KGPDC participates N N N N N in a newly discovered pathway for L-ascorbate fermenta- O R tion [30]. The OMP decarboxylase-catalyzed reaction is Hydantoinase Atrazine chlorohydrolase Adenosine deaminase metal-ion-independent [31] and must avoid a vinyl anion intermediate that is too unstable to exist; HPS [32] and NH2 H KGPDC [30] use Mg2þ to stabilize enediolate intermedi- NH2 O H2N N O N HO R ates in aldol condensation and decarboxylation, respec- O HN NH N H O N tively. The KGPDC from E. coli, like the OMP O H decarboxylases [33–36], is a dimer of identical polypep- O tides [12]. Structural superposition reveals conservation β Allantoinase -Aspartyl dipeptidase Cytosine deaminase of the polypeptide interface, conﬁrming divergent evolu- Current Opinion in Chemical Biology tion. (Conservation of the (b/a)8-barrel fold in a single domain protein is necessary but insufﬁcient to prove Substrates recognized by members of the amidohydrolase superfamily. evolution from a common progenitor.)

OMP decarboxylases and KGPDC share conserved active site functional groups: in OMP decarboxylase, these are and thus the resting state of the enzyme may only require involved in a hydrogen-bonding network that probably an hydroxide bound between the two divalent cations. destabilizes the substrate; in KGPDC, these provide ligands for the essential Mg2þ (Figure 5). Thus, divergent OMP decarboxylase suprafamily evolution can produce homologous enzymes that catalyse The ‘ribulose-phosphate binding barrel’ superfamily in different reactions with unrelated mechanisms. When the SCOP includes enzymes that catalyse mechanistically sequences of HPSs are used to query the databases, RPEs distinct reactions: are identiﬁed that share 28% sequence identity (Figure 5) (P Babbitt, W Novak and JA Gerlt, unpublished data). 1. Phosphoribosylformimino-5-aminoimidazole carboxa- The active site groups are not strictly conserved, but their mide ribonucleotide isomerase (HisA) and imidazole positions are conserved. RPE and HPS stabilize enedio- glycerolphosphate synthase (HisF) in histidine bio- late intermediates derived from ketose phosphates; how- synthesis. ever, the HPS-catalyzed reaction requires Mg2þ whereas 2. N-(50-phosphoribosyl)anthranilate isomerase (PRAI), the RPE-catalyzed reaction is independent of divalent indole-3-glycerol phosphate synthase (IPGS) and the metal ions [37,38]. Thus, divergent evolution produced a-subunit of tryptophan synthase (cleavage of indole homologous enzymes that stabilize enediolate intermedi- glycerol phosphate) in tryptophan biosynthesis. ates by distinct structural strategies. Resolution of 3. D-Ribulose 5-phosphate 3-epimerase (RPE) in the whether OMPDC, KGPDC, HPS and RPE are true pentose phosphate cycle. homologues of the ‘ribulose-phosphate binding’ enzymes 4. OMP decarboxylase in pyrimidine nucleotide bio- in histidine and tryptophan biosynthesis must await dis- synthesis. covery of ‘new’ sequences, perhaps of ‘new’ enzymes, These are clustered on the basis of a conserved phosphate that provide the ‘missing links’ which allow sequence binding motif at the ends of the seventh and eighth b- homologies to be established. strands. The recent conclusion that (b/a)8-barrels can be formed from (b/a)2N-subdomains has the potential of Aldolases invalidating the previous assumption that (b/a)8-barrels SCOP clusters several mechanistically distinct enzymes evolve en toto. So, the question remains whether the that catalyse aldol condensation reactions in an ‘aldolase’ members of this SCOP superfamily are true homologues. superfamily. Not all aldolases with the (b/a)8-barrel fold BLASTP searches of the databases provide both ‘new’ are clustered in this superfamily: HPS will be grouped in

(Figure 2 Legend) The reactions catalyzed by members of the enolase superfamily. www.current-opinion.com Current Opinion in Chemical Biology 2003, 7:252–264 258 Biocatalysis and biotransformation

Figure 4

His177 (a)Asp250 (b) His186 Asp243

His16 His12 His14 His18 His139

His156

Glu125 Lys102

His63 His189 His214 Glu125

Current Opinion in Chemical Biology

The active site motifs for members of the amidohydrolase superfamily. (a) DHO; (b) PTE homology protein; (c) cytosine deaminase; (d) human renal dipeptidase.

the ‘ribulose-phosphate binding barrel’ superfamily when 5. Type I dehydroquinate dehydratases (a Schiff base its structure is solved, and a family of class II aldolases is stabilizes an enolate intermediate in dehydration [42]. included in the ‘phosphoenol pyruvate/pyruvate domain’ superfamily. The diverse members of the ‘aldolase’ Whether these diverse enzymes are related is important in superfamily include: delineating the bounds and limits of divergent evolution. However, using the Dali algorithm, these show no more 1. Class I aldolases (a Schiff base with a lysine at the end structural homology to one another than to proteins in the of the sixth b-strand stabilizes an enediolate inter- ‘ribulose-phosphate binding barrel’ and the ‘phosphoenol- mediate, although in transaldolase the lysine is at pyruvate/pyruvate domain’ superfamilies. Although PSI- the end of the fourth b-strand) BLAST searches suggest some distant homology with 2. Some, but not all, class II aldolases (a divalent metal many (b/a)8-barrel proteins, most members of the super- ion stabilizes an enediolate intermediate). family do not show homology with one another or with 3. Porphobilinogen synthase (Schiff base intermediates other (b/a)8-barrel proteins. Until members are discovered with, perhaps, two lysine residues [39]). that show sequence homology, the assertion that these 4. Homologous heptulosonate [40], octulosonate [41] and diverse enzymes constitute an ‘aldolase’ superfamily is neuraminate phosphate synthases that use PEP as an intriguing but uncertain. enolpyruvate source for an aldol reaction (some are metal-dependent and others metal-independent) — Designed and directed evolution other enzymes that utilize enolpyruvate as intermedi- Attempts at directed evolution of enzymes with the ates are placed in the ‘phosphoenolpyruvate/pyruvate (b/a)8-barrel fold have been reported. Most altered the domain’ superfamily. substrate speciﬁcity of the progenitor, with the hope of

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Figure 5 biosynthetic pathway. Unfortunately, that claim was retracted, as subsequent characterization of the in vitro- (a) evolved PRAI was not possible due to its insolubility and inability to complement an auxotrophic strain, as origin- ally reported [44].

UMP Asp65∗ By contrast, exchange of substrate specificities between Arg215 functionally similar enzymes in the histidine and tryptophan biosynthetic pathways has been successful [45]. Both pathways include enzymes that catalyse Amadori Asp11 rearrangements of 10-aminonucleotides to yield aminoke- toses (HisA and PRAI). Despite the lack of discernible sequence identity, their structures reveal a shared (b/a)8- Lys62 barrel fold. Using a Trp auxotroph that lacks the gene encoding PRAI as the basis for a metabolic selection, Sterner and co-workers identified two active variants of Lys33 Asp60 HisA containing three and four amino acid substitutions in a library of random mutants. Assays using purified proteins demonstrated that both were able to catalyse, (b) albeit slowly, both the HisA and PRAI reactions. The seven substitutions were independently introduced into the HisA progenitor: only the Asp127Val mutant was L-Gulonate 6-P Asp67∗ sufficient to catalyse the PRAI reaction. Asp127 is located at the end of the fifth b-strand and is proximal to the Arg192 active site; in the absence of structures for the liganded progenitors and the evolved TrpF, a detailed structural explanation of the observed change in substrate specificity is unknown.

Asp11 Mg2+ Evolution of function in the enolase superfamily Lys64 One of our laboratories, together with Maxygen, Inc., is exploring the evolutionary potential of the (b/a)8-barrel in the enolase superfamily. The active sites of MLE II from Asp62 Pseudomonas sp. P51, the OSBS from E. coli, and the Glu33 L-Ala-D/L-Glu epimerase from E. coli contain lysine cat-

Current Opinion in Chemical Biology alysts at the ends of the second and sixth b-strands. In MLE, the lysine at the end of the second b-strand delivers a proton to the enolate intermediate formed The active site motifs for (a) the OMP decarboxylase from B. subtilis by addition of the remote carboxylate to the double bond; and (b) KGPDC from E. coli. in OSBS, the lysine at the end of the second b-strand both initiates the reaction by proton abstraction and protonates the hydroxide leaving group (D Klenchin, E Taylor- using an evolved enzyme with ‘improved’ properties Ringia, JA Gerlt and I Rayment, unpublished data); as catalysts in chemical applications. A few studies and in the epimerase the lysine residues catalyse a attempted alteration of the chemical reaction catalyzed two-base mechanism for 1,1-proton transfer. Neither by the progenitor. the MLE nor the epimerase catalyse the OSBS reaction. However, because these are homologues, all three Evolution of enzymes in histidine and tryptophan enzymes are derived from a common ancestor and a biosynthesis limited number of substitutions might allow interchange The most noteworthy report of directed evolution of the of activities. Design using the epimerase and combina- (b/a)8-barrel scaffold was reported by Fersht and co- torial evolution using the MLE both yielded proteins workers in 2000 [43]. The claim was made that alterations that catalyse the OSBS reaction and differ from their in the loops connecting several of the b-stands with the progenitor by a single amino acid substitution (DMZ. following a-helices allowed indole glycerol phosphate Schmidt, E Mundorff, PC Babbitt, J Minshull and JA synthase (IGPS) to efﬁciently catalyse the mechanisti- Gerlt, unpublished data). Structures are available for a cally distinct PRAI reaction, at the expense of the IGPS wild-type MLE, the epimerase and two OSBSs, so an reaction; both enzymes participate in the tryptophan explanation for the changes in activity is possible. In the www.current-opinion.com Current Opinion in Chemical Biology 2003, 7:252–264 260 Biocatalysis and biotransformation

design approach in which the structures of the epimerase genesis protocols in an effort to evolve and enhance the and the OSBS, both from E. coli, were compared, the substrate specificity of a given protein. In the first homologous aspartic acid at the end of the eighth instance, the inherent stereoselectivity of PTE for pairs b-strand of the epimerase was replaced with a glycine of chiral substrates has been enhanced, relaxed, and to generate sufficient volume so that the larger substrate inverted. For example, the wild type PTE catalyzes for OSBS might bind in the active site. In the combina- the hydrolysis of phenyl ethyl p-nitrophenyl phosphate torial approach with MLE, OSBS activity resulted from (1; Figures 6 and 7) with a 20-fold preference for the homologous substitution of a glutamic acid at the end of hydrolysis of (SP)-1. It was surmised from the X-ray crystal the eighth b-strand with a glycine. Both evolved proteins structure that the stereoselectivity was dictated by the had significant levels of OSBS activity and reduced levels orientation of the side chains that form the substrate of the progenitor activity. That two homologous substi- binding cavity within this enzyme. This notion was con- tutions in different progenitors results in a ‘new’ activity firmed by the mutation Gly60Ala. This single change emphasizes the functional plasticity of the (b/a)8-barrel within the active site altered the stereoselectivity from fold and suggests that nature may traverse different 20:1 to approximately 10 000:1 by specifically reducing mechanisms by targeting substitutions to the ends of the turnover rate for the initial slower (RP)-1 [46]. Con- the various b-strands, the positions that contribute the versely, if Ile106 is converted to glycine the stereoselec- catalytic functional groups. tivity is relaxed to the point where both enantiomers are turned over at essentially the same rate [47]. Finally, the Evolution of function in the amidohydrolase stereoselectivity was inverted to a ratio of 1:80 when only superfamily four mutations were made within the active site. These Specific members of the amidohydrolase superfamily changes specifically enhanced the rate of the initially have been subjected to rational and combinatorial muta- slower RP-enantiomer and diminished the rate of the

Figure 6

Substrate Intermediate Product

OMPDC O O O HN HN HN H+ − O N CO2 O N − O N H CO2

− O CO KGPDC CO2 H OH HO H CH2OH O O− O Mg2+ H OH H O HOH H HO H HO H HO H 2− 2− 2− CH2OPO3 CH2OPO3 CH2OPO3

O O HUPS H H H H CH2OH H OH CH2OH HO H O O− O Mg2+ H OH H O H H OH H OH H OH H OH 2- 2− 2− CH2OPO3 CH2OPO3 CH2OPO3

R5PE CH2OH CH2OH CH2OH O O− O H OH OH HO H H OH H OH H OH 2− 2− 2− CH2OPO3 CH2OPO3 CH2OPO3

Current Opinion in Chemical Biology

Substrates, intermediates and products of the reactions catalyzed by the members of the OMP decarboxylase suprafamily.

Current Opinion in Chemical Biology 2003, 7:252–264 www.current-opinion.com Evolution of function in (b/a)8-barrel enzymes Gerlt and Raushel 261

Figure 7

O O S EtO P O OP OEt OOP O O O

NO2 NO2 NO2 (Sp)-1 (Rp)-1 2

NH2 OCH3 NHCH3

N N N N N N

NNN NNN NNN 34 5

O SCH3 R O R

N N HN NH HN NH

NNN O O 6(S)-7 (R)-7

Current Opinion in Chemical Biology

Substrates used in the modification of substrate specificity within the amidohydrolase superfamily.

initially faster SP-enantiomer [47 ]. The specific mutation strates for which neither of the parents catalyzed the was Ile106Gly/Phe132Gly/His257Tyr/Ser308Gly. hydrolysis at a significant rate. These new substrates includedprometon(4), N-methylamino propazine (5) PTE has also been subjected to DNA shuffling in an andproetryn(6). There are only nine amino acid differ- attempt to improve the catalytic activity of the protein ences between AtzAandTriA. The most versatile new for the hydrolysis of methyl parathion [48].Themost mutant (active against the three new substrates) had six improved variant had an increase in catalytic activity of changes relative to AtzA and four changes relative to 25-fold over the wild-type enzyme for the methyl TriA. Unfortunately, the structure of this enzyme is parathion (2). This improved variant was shown to unknown and, thus, it is not yet possible to accurately contain seven amino acid differences relative to the map these alterations in three-dimensional space. wild-type enzyme. Of these changes, only the alteration of His257 to tyrosine appears to be located directly The HYD from Arthrobacter has been subjected to random within the active site. Therefore, alterations away from and saturation mutagenesis [50]. The native enzyme theactivesitecaninfluence the catalytic activity of preferentially hydrolyzes the D-enantiomer of a racemic PTE by structural or dynamic perturbations that are not mixture of D,L-methionine hydantoin (7) by a factor of fully understood. about 1.2. After two rounds of random and saturation mutagenesis, a single clone was produced that had a DNA shuffling has been used to direct the evolution of preference for the L-enantiomer by a factor of approxi- the atrazine chloro-hydrolase system (AtzA). In this mately 5. There was a single amino acid change relative to example, the gene encoding for atrazine chloro-hydro- the wild type enzyme. Ile95 was mutated to phenylala- lase was shuffled with the gene that encodes TriA, an nine. Unfortunately, the structure of this HYD is not enzyme that catalyzes the deamination of substituted known and thus the location of mutation is undetermined. melamines (3). The objective of this endeavour was to explore substrate plasticity using a small library of sub- Evolution of function in the aldolases strates bearing different leaving groups and a modified Despite the large number of aldolases in the sequence triazine core [49]. A single round of DNA shuffling was and structural databases, efforts to evolve their functions used and modified enzymes were isolated for three sub- have been restricted to the class I (Schiff base-forming) www.current-opinion.com Current Opinion in Chemical Biology 2003, 7:252–264 262 Biocatalysis and biotransformation

2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, References and recommended reading an enzyme in the Entner–Doudoroff pathway for glu- Papers of particular interest, published within the annual period of review, have been highlighted as: cose catabolism that yields pyruvate and D-glyceraldehyde 3-phosphate as products. KDPG aldolase has been of special interest used for synthetic applications because of its broad of outstanding interest substrate specificity. 1. Nagano N, Orengo CA, Thornton JM: One fold with many functions: the evolutionary relationships between TIM barrel families based on their sequences, structures and functions. Wong and co-workers [51] successfully evolved the aldo- J Mol Biol 2002, 321:741-765. A compendium of the structural features of (b/a) -barrels. lase from E. coli so that it could utilize unphosphorylated 8 substrates. Following an initial round of error-prone PCR, 2. Wierenga RK: The TIM-barrel fold: a versatile framework for efficient enzymes. FEBS Lett 2001, 492:193-198. mutants with relaxed specificity were identified by 3. Babbitt PC, Gerlt JA: Understanding enzyme superfamilies. screening. After two additional rounds of DNA shuffling, Chemistry as the fundamental determinant in the evolution of error-prone mutagenesis and shuffling, a variant was new catalytic activities. J Biol Chem 1997, 272:30591-30594. identified that was 600-fold more efficient that the 4. Lo Conte L, Brenner SE, Hubbard TJ, Chothia C, Murzin AG: SCOP wild-type progenitor in using the unphosphorylated sub- database in 2002: refinements accommodate structural strate, although the natural substrate was still preferred by genomics. Nucleic Acids Res 2002, 30:264-267. a factor of 85. This mutant with four substitutions also had 5. Copley RR, Bork P: Homology among (ba)8 barrels: implications for the evolution of metabolic pathways. J Mol Biol 2000, a relaxed enantiomeric preference for the glyceraldehyde 303:627-641. cosubstrate. Using the structure of the orthologueous 6. Lang D, Thoma R, Henn-Sax M, Sterner R, Wilmanns M: Structural KDPG aldolase from Pseudomonas putida, the substitu- evidence for evolution of the b/a barrel scaffold by gene tions were judged to be remote from the active site, duplication and fusion. Science 2000, 289:1546-1550. thereby preventing structure–function explanation for Structural evidence for the construction of HisA and HisF from a single (b/a)4-half barrel. the altered specificity. 7. Hocker B, Beismann-Driemeyer S, Hettwer S, Lustig A, Sterner R: Dissection of a (b/a)8-barrel enzyme into two folded halves. Toone and co-workers [52] used the novel approach of Nat Struct Biol 2001, 8:32-36. changing the position of the active site lysine to obtain a Biochemical evidence for the structural integrity and stability of the (b/a)4–half barrels in HisF. mutant that could utilize benzaldehyde as well as the 8. Wedekind JE, Poyner RR, Reed GH, Rayment I: Chelation of isosteric pyridine carboxaldehyde as a cosubstrate. serine 39 to Mg2þ latches a gate at the active site of enolase: 2þ Lys133, at the end of the sixth b-strand of the (b/a)8- structure of the bis(Mg ) complex of yeast enolase and the intermediate analog phosphonoacetohydroxamate at 2.1-A barrel, forms the Schiff base with the substrate. Screening resolution. Biochemistry 1994, 33:9333-9342. a library of random mutants identified the Thr161Lys 9. Eads JC, Ozturk D, Wexler TB, Grubmeyer C, Sacchettini JC: substitution as sufficient to confer the desired broadened A new function for a common fold: the crystal structure of specificity; this residue is at the end of the seventh quinolinic acid phosphoribosyltransferase. Structure 1997, b-strand. The Lys133Gln/Thr161Lys double mutant 5:47-58. was constructed to ‘require’ Schiff base formation at an 10. Hocker B, Schmidt S, Sterner R: A common evolutionary origin alternate position in the active site; this mutant retained of two elementary enzyme folds. FEBS Lett 2002, 510:133-135. modest activity on the natural substrates as well as the 11. Palmer DR, Garrett JB, Sharma V, Meganathan R, Babbitt PC, Gerlt JA: Unexpected divergence of enzyme function and sequence: desired enhanced activity with benzaldehyde. This phe- ‘‘N-acylamino acid racemase’’ is o-succinylbenzoate synthase. notype was attributed to an alteration of the size and Biochemistry 1999, 38:4252-4258. polarity of the binding site for the aldehyde cosubstrate. 12. Wise E, Yew WS, Babbitt PC, Gerlt JA, Rayment I: Homologous (beta/alpha)8-barrel enzymes that catalyze unrelated reactions: orotidine 50-monophosphate decarboxylase and Conclusions 3-keto-L-gulonate 6-phosphate decarboxylase. Biochemistry The recent studies of (b/a)8-barrel-containing enzymes 2002, 41:3861-3869. Structural evidence which confirms that divergent evolution can produce summarized in this review highlight the range of natural enzymes that catalyse different reactions with different mechanisms functional divergence. The problem of determining the using conserved active site functional groups. OMP decarboxylase catalyzes a metal-independent reaction that must avoid a vinyl anion number of natural progenitors remains a challenge that intermediate; KGPDC catalyzes a Mg2þ-dependent reaction that stabi- will probably be solved as ongoing genome and structural lizes an enediolate anion intermediate. genomics projects add to the sequence and structure 13. Gerlt JA, Babbitt PC: Divergent evolution of enzymatic function: databases. If this number is small (one?), we predict that mechanistically diverse superfamilies and functionally distinct both the design and discovery of enzymes that catalyse suprafamilies. Annu Rev Biochem 2001, 70:209-246. novel reactions on unnatural substrates will become fea- 14. Babbitt PC, Mrachko GT, Hasson MS, Huisman GW, Kolter R, Ringe D, Petsko GA, Kenyon GL, Gerlt JA: A functionally diverse sible and, perhaps, routine. enzyme superfamily that abstracts the alpha protons of carboxylic acids. Science 1995, 267:1159-1161. 15. Schmidt DM, Hubbard BK, Gerlt JA: Evolution of enzymatic Acknowledgements activities in the enolase superfamily: functional assignment of The research in the authors’ laboratories is funded by: JAG, NIH GM-52594 unknown proteins in Bacillus subtilis and Escherichia coli as and NIH GM-58506; FMR., NIH GM-33894 and Office of Naval Research L-Ala-D/L-Glu epimerases. Biochemistry 2001, (N00014-99-0235). 40:15707-15715.

Current Opinion in Chemical Biology 2003, 7:252–264 www.current-opinion.com Evolution of function in (b/a)8-barrel enzymes Gerlt and Raushel 263

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Biochemistry specificity of phosphotriesterase. Biochemistry 2001, 2001, 40:2712-2722. 40:1325-1331. 30. Yew WS, Gerlt JA: Utilization of L-ascorbate by Escherichia coli 47. Chen-Goodspeed M, Sogorb MA, Wu F, Raushel FM: K-12: assignments of functions to products of the yjf-sga and Enhancement, relaxation, and reversal of the yia-sgb operons. J Bacteriol 2002, 184:302-306. stereoselectivity for phosphotriesterase by rational evolution of active site residues. Biochemistry 2001, 40:1332-1339. 31. Miller BG, Smiley JA, Short SA, Wolfenden R: Activity of yeast The substrate binding pockets for PTE were manipulated in a rational orotidine-50-phosphate decarboxylase in the absence of manner to alter the stereoselectivity. With only 4–5 amino acid changes metals. J Biol Chem 1999, 274:23841-23843. within the active site, the stereoselectivity of the wild-type enzyme can be manipulated a factor of one-million-fold. 32. Sahm H, Schutte H, Kula MR: Purification and properties of 3- hexulosephosphate synthase from Methylomonas M15. 48. Cho CM, Mulchandani A, Chen W: Bacterial cell surface display Eur J Biochem 1976, 66:591-596. of organophosphorus hydrolase for selective screening of www.current-opinion.com Current Opinion in Chemical Biology 2003, 7:252–264 264 Biocatalysis and biotransformation

improved hydrolysis of organophosphate nerve agents. 51. Fong S, Machajewski TD, Mak CC, Wong C: Directed evolution of Appl Environ Microbiol 2002, 68:2026-2030. D-2-keto-3-deoxy-6-phosphogluconate aldolase to new variants for the efficient synthesis of D- and L-sugars. 49. Raillard S, Krebber A, Chen Y, Ness JE, Bermudez E, Trinidad R, Chem Biol 2000, 7:873-883. Fullem R, Davis C, Welch M, Seffernick J et al.: Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes. Chem Biol 2001, 8:891-898. 52. Wymer N, Buchanan LV, Henderson D, Mehta N, Botting CH, The paper is an outstanding example for the creation of novel enzyme Pocivavsek L, Fierke CA, Toone EJ, Naismith JH: Directed activities through gene shuffling methodology and characterization of evolution of a new catalytic site in 2-keto-3-deoxy-6- substrate profiles with a small compound library. phosphogluconate aldolase from Escherichia coli. Structure 2001, 9:1-9. 50. May O, Nguyen PT, Arnold FH: Inverting enantioselectivity by The position of the active site lysine residue in the class I KGPD aldolase directed evolution of hydantoinase for improved production of was moved from the sixth to the seventh b-strand to give a mutant with L-methionine. Nat Biotechnol 2000, 18:317-320. enhanced substrate specificity.

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