Structure of Bacterial Glutathione-S-Transferase Maleyl Pyruvate Isomerase and Implications for Mechanism of Isomerisation

J. Mol. Biol. (2008) 384, 165–177

doi:10.1016/j.jmb.2008.09.028

Available online at www.sciencedirect.com

Structure of Bacterial Glutathione-S-Transferase Maleyl Pyruvate Isomerase and Implications for Mechanism of Isomerisation May Marsh 1 , Deborah K. Shoemark 1 , Alyssa Jacob 1 , Charles Robinson 1 , Brent Cahill 1 , Ning-Yi Zhou 2 , Peter A. Williams 2 and Andrea T. Hadfield 1 ⁎ 1

Department of Biochemistry and Centre for Molecular Recognition, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK 2

Department of Biological Sciences, University of Wales, Bangor, UK Received 1 July 2008; received in revised form 1 September 2008; accepted 9 September 2008 Available online 19 September 2008

Maleyl pyruvate isomerase (MPI) is a bacterial glutathione S-transferase (GST) from the pathway for degradation of naphthalene via gentisate that enables the bacterium Ralstonia to use polyaromatic hydrocarbons as a sole carbon source. Genome sequencing projects have revealed the presence of large numbers of GSTs in bacterial genomes, often located within gene clusters encoding the degradation of different aromatic compounds. This structure is therefore an example of this under-represented class of enzymes. Unlike many glutathione transferases, the reaction catalysed by MPI is an isomerisation of an aromatic ring breakdown product, and glutathione is a true cofactor rather than a substrate in the reaction. We have solved the structure of the enzyme in complex with dicarboxyethyl glutathione, an analogue of a proposed reaction intermediate, at a resolution of 1.3 Å. The structure provides direct evidence that the glutathione thiolate attacks the substrate in the C2 position, with the terminal carboxylate buried at the base of the active site cleft. Our structures suggest that the C1–C2 bond remains fixed so when rotation occurs around the C2–C3 bond the atoms from C4 onwards actually move. We identified a conserved arginine that is likely to stabilize the enolate form of the substrate during the isomerisation. Arginines at either side of the active site cleft can interact with the end of the substrate/product and preferentially stabilise the product. MPI has significant sequence similarity to maleylacetoacetate isomerase (MAAI), which performs an analogous reaction in the catabolism of phenylalanine and tyrosine. The proposed mechanism therefore has relevance to the MAAIs. Significantly, whilst the overall sequence identity is 40% only one of the five residues from the Zeta motif in the active site is conserved. We reexamined the roles of the residues in the active site of both enzymes and the Zeta motif itself. © 2008 Elsevier Ltd. All rights reserved.

Edited by R. Huber

Keywords: glutathione transferase Zeta; isomerisation; biodegradation; maleylacetoacetate isomerase; enzyme–substrate complex X-ray structure

Introduction Microbes found in natural waters and soils have between them a very broad ability to utilise naturally *Corresponding author. E-mail address: [email protected]. Abbreviations used: MAAI, maleylacetoacetate isomerase; GST, glutathione S-transferase; MPI, maleyl pyruvate isomerase, maleyl pyruvate isomerase.

occurring compounds as sources of carbon and energy, enabling them to recycle the fixed, organic carbon back into harmless biomass and carbon dioxide. The advent of the modern chemical industry has resulted in the release into the environment of huge amounts of novel organic compounds. Bacteria appear able to adapt their pre-existing catabolic breadth to enable them to attack and degrade many of these novel xenobiotic compounds. Ralstonia is one such microbe that has been isolated from oilfields in Venezuela, municipal sewage sludge and

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Maleyl Pyruvate Isomerase: Mechanism of a ζ-GST

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Fig. 1. The pathways for catabolism of (a) naphthalene via gentisate (b) tyrosine via homogentisate.

petroleum-contaminated soil in the USA, and soil at an industrial waste deposit in Germany. Different strains can use chlorobenzenes, bromobenzenes and naphthalene as their sole carbon source. Naphthalene, a fused ring bicyclic aromatic hydrocarbon commonly found in crude oil and oil products, is a model for understanding the properties of a large class of environmentally prevalent polycyclic aromatic hydrocarbons, some of which are strong human carcinogens. In the ”classical” route for bacterial naphthalene catabolism, naphthalene is converted to salicylate (2hydroxybenzoate), which is then further broken down to central metabolites with catechol as an intermediate: the enzymes required for the two sequences of reactions (naphthalene to salicylate and salicylate to central metabolites via catechol) are encoded on physically separate operons. In Ralstonia U2, salicylate is also formed from naphthalene but is then metabolised through the gentisate (2,5-dihydroxybenzoate) pathway and the entire set of catabolic genes is in a single operon (Fig. 1).1,2 The bacterial gentisate pathway is also used for a variety of other pollutant xenobiotics and is chemically analogous to the mammalian homogentisate (2,5-dihydroxyphenylacetate) pathway, through which the amino acids phenylalanine and tyrosine are cata>bolised to central metabolites. There are a number of genetically inherited mutations associated with the enzymes in the human homogentisate pathway, including phenylketonuria, alkaptonuria and hereditary tyrosinemia type 1. Maleyl pyruvate isomerase (MPI) is a 212 amino acid protein (23 kDa) from Ralstonia U2 that catalyses

the third committed step in the degradation of salicylate to the metabolites pyruvate and fumarate via gentisate. Sequence analysis shows that it is a member of the glutathione S-transferase (GST) Zeta family.3 In contrast to most classes of GST, the members of the Zeta class have poor GSH-conjugating activity with standard or conventional GST substrates such as 1-chloro-2,4-dinitrobenzene, ethacrynic acid and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. Their two primary activities are the cis-trans isomerisation of maleate derivatives, including maleyl acetoacetate and maleylpyruvate, and the dehalogenation of α-haloacids such as dichloroacetic acid. Both reactions require glutathione but, unlike other classes of glutathione transferase, the conjugation is reversible and the glutathione is not consumed (it is a true cofactor and not a co-substrate). For the isomerisation reaction to occur, a double bond between C2 and C3 of the substrate has to be broken, which suggests attack by the glutathione on either the C2 or C3 carbon. The microbial GSTs have been ignored for a long time due to the very low activity with standard GST substrates of mammalian and plant enzymes that are detected in micro-organisms. However, genome sequencing projects have revealed the presence of large numbers of GSTs of unknown function in bacterial and yeast genomes (as many as 30 in a single organism).4,5 Bacterial GST genes are often located within gene clusters encoding the degradation of different aromatic compounds such as biphenyls, polycyclic aromatic hydrocarbons, 2,4,5-trichlorophenoxyacetate, and dibenzodioxin,1,6-9 which suggests an important role of many such proteins in the

Fig. 2. (a) A cartoon representation of the MPI dimer, with the A subunit shown in purple and the B subunit shown in green. (b) A cartoon representation of the A subunit as above, showing glutathione in bonds representation. The view is looking down on the substrate-binding cleft. Carbon colours are the same as in (a). (c), The glutathione (GSH) binding site. Residues interacting with the glutathione are shown in ball-and-stick representation. d, The dicarboxyethyl-glutathione binding site. Residues interacting with DCEG are show in bonds representation. Carbon colours are the same as in (a). (e), A stereo view of 2Fo – Fc electron density map showing electron density for dicarboxyethyl-glutathione, contoured at 1.0σ.

Maleyl Pyruvate Isomerase: Mechanism of a ζ-GST

Fig. 2 (legend on previous page)

167

168 dehalogenation, detoxification and mineralisation of environmental pollutants and their degradation products. Whilst eukaryotic GSTs are well represented structurally, there are currently few structures of the bacterial enzymes in the Protein Data Bank. MPI shows significant sequence similarity to maleylacetoacetate isomerase (MAAI), which performs an analogous reaction in the catabolism of phenylalanine and tyrosine (Fig. 1). Two eukaryotic maleylacetoacetate isomerase structures have been pub- lished, from human10 and mouse-ear cress (Arabadopsis Thaliana).11,12 The sequences of both enzymes share 40% identity with MPI. The distinctive features of the Zeta class GSTs include the motif S14S15C16XWRVRIAL identified by Board et al. in 1997, where the subscript numbers refer to the sequence position in human MAAI. This and subsequent publications mistakenly refer to the sequence SSCXWRVIAL, which has a sequence deletion of a conserved arginine that is always found between the underlined residues. The motif was further described as LYSYW R / L S 11 SC 13 SX R / K VRIAL through an analysis that includes tetrachlorohydroquinone dehalogenase, where the numbers refer to the position in the tetrachlorohydroquinone dehalogenase sequence.13 However, six of these 11 residues are not conserved in MPI, including some suggested as important for substrate binding and catalysis. The N-terminal sequence of MPI in this fingerprint region is S9G10T11S12HRLRIAL. The Zeta class of GSTs is known to be responsible for metabolising dichloroacetic acid and other α-halo acids, including dichloroacetic acid.14-17 A structure for MAAI, with sulphate observed in the active site, and mutational data led to the proposal of a mechanism10-12 whereby the serine at the beginning of the motif stabilises the thiolate ion of glutathione, which can therefore undergo nucleophilic addition to the C2 of maleyl acetoacetate. An enolate is thus formed, and rotation can take place around the resulting single bond between C2 and C3. Glutathione is eliminated, regenerating the enzyme and co-factor, along with the product fumarylacetoacetate. In this work, we have determined the structure of an analogue of the covalent adduct with a single bond between C2 and C3. Analysis of this structure and comparison of the structures of maleyl acetotacetate isomerase and MPI, which catalyse isomerisation reactions on similar substrates (Fig. 1) gives insights into the mechanism. We have used the sequence analysis and alignment tools available at the European Bioinformatics Institute to reassess the consensus sequence of this family of enzymes.

Results Overall description Examination of the crystal contacts shows that MPI crystallizes with a dimer in the crystallographic asymmetric unit (Fig. 2a), which corresponds to the oligomeric molecular mass observed during gel fil-

Maleyl Pyruvate Isomerase: Mechanism of a ζ-GST

tration. The A and B subunits are very similar, with an RMS displacement for Cα atoms of 0.1 Å, the and the following discussions therefore concentrates on the A subunit. The MPI fold is similar to other Zeta class GST family members (Fig. 2b). The N-terminal domain comprises a four-stranded β-sheet flanked on the outside by two short α-helices and on the inside by two longer α-helices, and is connected to the C-terminal domain by a linker region. The C-terminal domain is entirely α-helical. Helices α4 and α5 in the C-terminal domain are largely responsible for dimerisation, which buries 2260 Å2, compared to a total surface area per subunit of 12,360 Å2, with additional contacts generated through the packing of short helix α3 in the N-terminal domain against the N terminus of helix α4 in the opposite C-terminal domain. Mammalian GST structures have been described as having an intersubunit lock and key interaction, with the key being provided by a hydrophobic residue.18 In MPI, the equivalent residue is glutamine, which hydrogen bonds with the opposite subunit via residue Glu102, which also participates in a crosssubunit hydrogen bond with the glutathione as described below. Polekhina et al. showed that the equivalent residue in MAAI is methionine (Met 56humB), and appears less intimately involved in dimerisation of the human MAAI than in other families of GSTs.10 Glutathione binding (G-site) The glutathione lies at the bottom of a deep crevice, close to the dimerisation interface. Electron density for the glutathione was clearly visible in the first molecular replacement electron density maps before it was modelled. The glutathione binds in an extended conformation, orientated approximately parallel with the twofold axis of the protein (Fig. 2) and forms extensive interactions with the protein (Fig. 2c). We observed that glutathione binding includes residues from both subunits. The glycyl moiety interacts with Asn108, the main chain N of Arg 110 and NE2 from His 38. The cysteinyl moiety in the centre of GSH makes interactions characteristic of the glutathione transferases including the carbonyl O of Val52, with Pro53 arranged in the cis conformation. In the holo complex, the cysteinyl sulphur lies within hydrogen bonding distance of the hydroxyl oxygens of Ser9 and Thr11, and the epsilon nitrogen of His104. These interactions hold it at the N terminus of helix α1. The γ-glutamyl moiety interacts with Gln64, which lies in the generously allowed region of the Ramachandran plot, which is commonly observed in GSTs (φ = 74°, ψ = 96°). However, in contrast to other Zeta class structures, there is a direct hydrogen bond between Glu102 of the neighbouring subunit and the amino group of this moiety, as observed in the Mu class of GSTs.19 Dicarboxyethyl glutathione binding The interactions of the glutathione portion of the dicarboxyethyl glutathione (DCEG) complex are the

Maleyl Pyruvate Isomerase: Mechanism of a ζ-GST

same as in the holo complex,with the exception of the sulphur of the cysteinyl moiety, which takes an alternative rotamer conformation pointing further out into the active site crevice (Fig. 2d and e). This sulphur is covalently attached to the dicarboxyethyl group in the C2 position. The C1 carbon has its carboxyl oxygens buried at the inner end of the active site cleft. One of the oxygens is hydrogen bonded to the main chain nitrogen of Gly10, a residue within the signature motif, and the other to the epsilon nitrogen of His104. One of the carboxyl oxygens of the second carboxylate, at C4, makes a hydrogen bond with the backbone nitrogen of Arg109, and the other with the highly conserved Arg176 (Fig. 2d and e). Building maleyl and fumaryl pyruvate in the active site (H-site) The carboxy groups of DCEG have the same separation as those in maleate, a component of the maleyl pyruvate substrate. Models of maleyl pyruvate and fumaryl pyruvate have been built into the active site, based on the observed position of the dicarboxyethyl moiety (Fig. 3). The maleyl group was positioned by aligning its C2–C3 bond approximately along the C2–C3 bond in DCEG, while preserving the interactions observed in DCEG of the C1 carboxylate with the main chain nitrogen on Gly10 and His104N. The residue Arg176ral, which is widely conserved in Zeta GSTs is not very well ordered in either the holo or DCEG structures. In the DCEG complex it is observed in two different conformations, in one of which it interacts with the pyruvyl

169 moiety of the modelled substrate. The other conformation is appropriate to interact with the O4 hydroxyl of maleyl pyruvate. Further inspection of the active site reveals two further positively charged residues, Arg8 and Arg109. The maleyl pyruvate can be built in such a way that both Arg8 and Arg109, as observed in the DCEG structure, interact with the pyruvyl moiety and favourable interactions are maintained around the carboxylate and the C2 at the site of catalysis. In this position, it is possible to flip the maleyl pyruvate moiety around the C2–C3 bond to create a fumaryl pyruvate molecule where the tight binding of the terminal carboxylate, which is relatively buried in the active site, is maintained. Arg 8 is pointing out of the active site in holo-MPI but points in towards the active site in the DCEG structure, hydrogen bonding to a water network that is replaced in the substrate/product models. A further model of a possible reaction intermediate was built, again using the DCEG structure as a guide. This putative intermediate has the covalent character of the analogue, and the enolate oxygen attached to carbon C4 takes the position of the carboxylate oxygen of DCEG, which hydrogen bonds with the main chain nitrogen of Arg109. Comparison of overall fold to MAAI human, cress The structures of human 10 and cress 11 MAAI were overlaid on the A subunit of MPI for comparison (Fig. 4). The overall architecture of MPI is very similar to that of MAAI, consistent with the

Fig. 3. A stereo view of the active site of the MPI–DCEG complex (purple sticks, DCEG in cyan), with models for maleyl pyruvate (MAL, green) and fumaryl pyruvate (FUM, blue) overlaid.

170

Maleyl Pyruvate Isomerase: Mechanism of a ζ-GST

Fig. 4. (a) The Cα trace of A. thaliana MAAI (1E6B, forest green), in the absence of glutathione with conserved arginines 16 and 181 (shown in ball and stick representation) and residues 113 and 133, between which the structure is disordered, circled. Overlaid is a Cα trace of holo-Ralstonia MPI with glutathione (2C1R, pink) and the conserved arginines 8 and 176 shown in ball and stick representation. (b) The Cα trace of holo-human MAAI (1FW1, blue) with glutathione and the conserved arginines 13 and 175, and a sulphate observed in the crystal structure, shown in ball and stick representation overlaid on the Cα trace of Ralstonia MPI (2V6K, magenta) with substrate analogue dicarboxyethyl glutathione and the conserved arginines 8 and 176 shown in ball and stick representation, viewed at approximately 90° rotation from that in (a). (c) Atereo view of the active site of the MPI–DCEG complex (purple sticks, DCEG in cyan), with MAAI-sulphate complex overlaid (blue sticks).

level of sequence identity being ∼40%. On the basis of the fold, MPI belongs to the Zeta family of GSTs. Helix 4a is one turn longer in MPI, making the loop

between helix 4 and helix 5 more extended. The structures of MAAI from cress and mouse were solved in the absence of glutathione and the region of

Maleyl Pyruvate Isomerase: Mechanism of a ζ-GST

the structure from helix 4a, to the centre of helix 5, is disordered in the cress structure (residues 113–133) (Fig. 4a) and the B subunit of the mouse structure (B116–B124), whereas the A subunit of the mouse structure is disordered on the opposite side of the glutathione-binding cleft (A37–A57). The MPI holo structure has glutathione bound, and apart from that appears to have just simple solvent molecules (water) in the active site. The whole protein chain is ordered apart from the N-terminal His tag. The glycyl moiety of glutathione in Ralstonia MPI and human MAAI makes hydrogen bonds with residues corresponding to the disordered part of MAAI in cress and mouse MAAI, and are presumably responsible for those parts of the structure becoming ordered in the presence of glutathione. Comparing the MPI holo structure with the human MAAI holo structure (Fig. 4b and c), there are differences in the orientation of conserved residues Arg175BhumB (176BralB) and Arg13BhumB (8BralB), which in

171 human MAAI are observed to make hydrogen bonds with a sulphate ion that binds in the active site. The structure of MPI in the presence of DCEG represents an analogue of the substrate, indeed, of a reaction intermediate where the C2–C3 bond is single. Comparison of the active sites of MPIDCEG and human MAAI shows that the sulphate is in a similar position to the terminal carboxylate of the substrate analogue DCEG (Fig. 4c) rather than the acetate at the far end of the substrate of MAAI, as modelled by Polekhina et al. 10 The glutamine conserved in MAAIs can be seen superimposing well on the position of His104. Conserved Ser14hum (9ral) points out of the active site, and was argued by Polekhina et al. not to be involved in catalysis on this basis,10 whereas it points into the active site in both the holo MPI and the substrate complex MPI structures, and is implicated in thiolate stabilisation and substrate binding through a simple change in rotamer. This is the only residue in the active site

Fig. 5. Possible steps in mechanism, in structures and models. (a) The active site cleft shown in sticks representation, holo-Ralstonia MPI with glutathione crystal structure. (b) A model of substrate maleyl pyruvate in the active site. (c) A model of the covalent intermediate based on the DCEG structure. d, a model of the product fumaryl pyruvate in the active site.

172 from the Zeta motif that is conserved between MAAI and MPI. Ser15hum is replaced by glycine in MPI and Cys16hum in MAAI is replaced by threonine in MPI. Maleyl acetoacetate, the substrate for MAAI, is the length of one carbon bond longer than maleyl pyruvate. The substrate interactions proposed for MPI would also work for MAAI, with the exception of Arg109 which is Leu in MAAI. There is a lysine present (Lys120, Fig. 4c) one turn further along in the α-helix in human MAAI, where there is no Arg109 that might have been able to form similar interactions with the terminal carboxylate of MAAI. A positively charged residue is present in an equivalent location in many of the MAAI sequences.

Discussion Catalysis The MPI and MAAI structures provide a series of snapshots of the enzyme at different stages through a reaction cycle. Taking the structures together, a picture builds up with an active site binding loop disordered in the apo form of the enzyme becoming ordered in the presence of glutathione. Finally, the long charged surface residues that are somewhat disordered in the absence of substrate become locked down in the active site when the substrate binds. The cress and mouse MAAI structures represent the apo form of the enzyme, awaiting its cofactor. The holo structure represents the enzyme ready for action, with glutathione bound, but the active site cleft open with arginine side chains not well ordered and pointing towards the solvent (Fig. 5a). The reactive thiol is buried, stabilised in the thiolate form by residues in the active site; Ser9 and Thr11 within the Zeta family motif and His104. This positioning of the equivalent serine has not been observed in the MAAI structures,10,12 but has been postulated. A role in catalysis for this serine is confirmed by various mutagenic studies on MAAI enzymes.12,20,21 The conformation of the substrate analogue DCEG is not restricted and it could bind either with an

Maleyl Pyruvate Isomerase: Mechanism of a ζ-GST

aliphatic carbon and a carboxylate facing towards the interior of the protein from the covalent linkage, which would represent attack on C3, or with a single carboxylate in the interior position compared to the covalent linkage, as is observed. The position in which the dicarboxyethyl moiety binds therefore gives direct structural evidence for attack of the glutathione on the C2 carbon, which was suggested in 1978 by Seltzer on the basis of solution studies of the coenzyme-catalysed isomerisation of maleylpyruvate and acetoacetate.22 The structures suggests a possible mechanism whereby maleyl pyruvate enters the active site and starts to be oriented by the hydrogen bonding of the terminal carboxylate to the main chain nitrogen of Gly10 within the signature motif, and the side chain of His104. This fixes the end of the substrate and locates the C2 carbon in a suitable position to be attacked by the GSH thiolate (Fig. 5b). The thiolate ion could be generated by proton abstraction by His104, as well as stabilised by Thr11 and Ser9. The thiolate attacks C2, and the double bond C2–C3 becomes single, as suggested by earlier studies of the mechanism.12,22 Rotation is permitted now that the bond is single. Rotation by 180° of the pyruvyl moiety in the exposed end of the active site around the C2–C3 bond brings the molecule into the trans isomer, fumarylpyruvate, rather than the rotation of the C2–C1 portion as proposed by Seltzer (Fig. 6).22 Apart from confirming the position of chemical attack in the catalysis, the structures shed light on a possible explanation for the isomerisation itself. The positioning of positively charged arginine residues at either side of the active site cleft in the vicinity of the pyruvyl moiety of the substrate/product allow for favourable interactions both with the substrate and product, i.e. in both cis and trans conformations. The reaction mechanism proposed (Fig. 6) would result in an accumulation of negative charge on the enolate oxygen attached to C4 as the double bond between C2 and C3 becomes a single bond. Arg176 is in a position where one rotamer would allow stabilisation of this accumulating negative charge, which develops as electrons are pushed through upon

Fig. 6. A scheme for a possible mechanism.

Maleyl Pyruvate Isomerase: Mechanism of a ζ-GST

attack at position C2 (Fig. 6iii). This arginine is very highly conserved in an alignment of the 200 sequences most similar to MPI, supporting a role in catalysis, not simply in substrate binding as previously proposed.10 Maleyl pyruvate is in the less energetically favourable cis conformation. Following Michael addition of the thiolate at C2, the molecule has rotation around the C2–C3 bond, which is a single bond, and can therefore explore different conformations. The covalent intermediate model (Fig. 5c) represents a possible step along the reaction pathway where this negatively charged oxygen can be stabilised by the equivalent hydrogen bond to that observed in the DCEG structure with the main chain nitrogen of Arg109. After further rotation in the same direction, it reaches the trans conformation of fumaryl pyruvate, which is energetically favoured (Fig. 5d).23 In addition to this extra stability, the organisation of the active site around the substratebinding site suggests that there may be more stabilising interactions available for the trans isomer rather than the cis isomer, promoting the preferential release of fumaryl pyruvate rather than maleyl pyruvate after attack by the glutathione thiolate on C2. These interactions could include a hydrogen bond between the C4 hydroxyl and N3 of the glutathione, and bidentate hydrogen bonds with the conserved Arg8 and Arg176, and further monodentate hydrogen bonding with Arg109. Kiefer and Copley drew comparisons with the mechanism of tetrachlorohydroquinone dehalogenase.24 They suggested that underlying mechanistic similarities between the dehalogenase and isomerase reactions could include: (a) protonation of the substrate by an active site acid and removal of a hydroxyl proton by an active site base to “prepare” the electrophilic substrate for attack by glutathione; and (b) facilitation of the nucleophilic attack of glutathione upon an “enone” substrate by ionization of glutathione at the active site and possibly electrophilic assistance at the carbonyl group. There is no evidence in the active sites of MPI or MAAI for a side chain base to accept a proton from the enol form, or a side chain acid to protonate C5. Whilst the enol form of the substrate is likely to be the most stable form in solution, it is able to tautomerize to produce the enone, which would activate the substrate for nucleophilic attack by glutathione. It is possible that the active site with its positively charged arginines is able to bind the enone form selectively directly from solution. Early work on the silver-catalysed isomerisation of maleylacetoacetate to fumarylacetoacetate suggested that the ionisation of the maleyl terminal carboxyl group, and the enolisation of the adjacent carbonyl are of great importance in promoting the isomerisation.23 The enzymes MAAI and MPI both provide stabilisation for an ionised carboxyl group. In the case of MPI, this is accomplished by a main chain hydrogen bond with Ser9 and a side chain hydrogen bond with His104. A protonated and therefore positively charged His104, which would result from abstracting a proton from the thiolate, would be particularly effective in this role.

173 The Zeta family motif The Zeta family of GSTs is a relatively recent addition to the superfamily of GSTs. The original classification based on the limited number of (eukaryotic) available maleyl acetoacetate isomerase sequences identified a fingerprint motif of SSCxWRVIAL,3 which was extended by Anandarajah et al. to LYSYWR/LSSCSXR/KVRIAL.13 The analogous MPI in the catabolism of salicylate via gentisate has 40% sequence identity. Whilst the overall architecture of MPI and MAAI is very similar, only one residue in the first five of the active site Zeta family motif, those commonly associated with catalytic functions, is preserved in MPI. This is serine 9ralB, and this is in a position to hydrogen bond with the cysteinyl sulphur ion and maintain a thiolate ion, a role proposed for the serine in the Zeta and in many other classes of glutathione transferase, including Theta, Delta, insect Epsilon, plant Phi and Tau. The sequence in the active site region of the motif for Ralstonia U2 MPI is SGTSHRLRIAL, where matches with the established Zeta family motif are shown in bold. Alignment of more than 200 sequences N 40% identical to the MPI sequence reveals differences between prokaryotic organisms and eukaryotic organisms in this class of glutathione transferases (Fig. 7). The consensus sequence in the eukaryotic members of the family is SSCxWRVRIAL (the second arginine, shown here in italics, was omitted in the original description and the sequence motif has been widely misquoted), whereas the prokaryotic members of the family have the consensus RSSASYRVRIAL, where the residues shown in bold are N85% conserved. The underlined residues are all small polar/ non-polar residues (A,G, S,T) and the residue in italics is most frequently Y but is occasionally replaced by aromatic residues F, W or H. This consensus includes a highly conserved arginine at the N terminus of the conserved region. The importance of these highly conserved residues corresponds to the roles of the side chains based on the observations from the MPI structures and mutational studies in MAAI. Arginine 8, at the beginning of the motif, appears likely to have an important role in substrate recognition. A small group of enzymes, including MPI/MAAI from Escherichia coli, Klebsiella pneumoniae MPI, Salmonella enterica, Salmonella typhimurium, Ralstonia solanacearum and Pseudomonas aeruginosa all have asparagine in the position equivalent to Arg8 but they all have arginine in position 34Ral, which by inspection of the structure would lie adjacent and could have a functional group in same place. Serine 9, which is also strongly conserved, has a proposed catalytic role both in thiolate stabilisation, in this work for MPI and as discussed, and demonstrated by site-directed mutation, for MAAI by Lapthorn and colleagues.12 This serine is also the equivalent of the catalytic serine in the theta and delta classes of GSTs. The third, fourth and fifth positions in this motif are a combination of Ser, Thr and Ala, and the sixth position is Tyr, Phe or His. These four less highly

174

Maleyl Pyruvate Isomerase: Mechanism of a ζ-GST

Fig. 7. Excerpts from an alignment of 200 sequences retrieved from UNIPROT. Numbering across the top corresponds to positions in the Ralstonia U2 MPI sequence. Q89Y46_BRAJA: Bradyrhizobium japonicum MAAI, Q3S4B4_9BURK Polaromonas naphthalenivorans (strain CJ2) MPI:, O86043_9RALS, Ralstonia U2 MPI, Q5EXK2_KLEPN Klebsiella pneumoniae MPI, Q5K5T6_ECOLI Escherichia Coli putative GST, GSTZ1_ARATH Arabadopsis Thaliana MAAI, MAAI_HUMAN: human MAAI (GSTZ1). The catalytic serine is shown in yellow with a black background. The position where some sequences have Cys in the active site (grey background) is indicated by ▌below, with the partner residue sequence position marked with ▌. A filled diamond (♦) indicates the position of the proposed catalytic arginine, shown with a yellow background. The proposed substrate-binding arginine and its possible alternative are shown with a green background.

conserved residues form the first turn of helix α1. The hydrophobic residue at the sixth position and the subsequent five highly conserved residues form the core of the molecule at the N terminus of helix 1, which is buried and forms the major part of the interface between the N and C terminal domains of the subunit. Thr11 also lies within hydrogen bonding distance of the glutathione sulphur in MPI. This residue (fourth position in the motif) replaces the conserved cysteine in the Zeta family motif in currently characterised MAAIs. In N 140 sequences where this cysteine is replaced by a small, usually polar side chain as described above (Ser, Thr, Ala), His104 replaces a conserved glutamine. This histidine is present in a wide range of prokaryotic sequences, where the cysteine in the proposed Zeta motif is missing. In this set of enzymes, it is possible that the histidine acts as a base, abstracting a proton from the glutathione thiol group to generate a negatively charged thiolate ion. This is in an appropriate position to be stabilised by hydrogen bonds with the hydroxyl groups of Ser9 and Thr11. The presence of the thiolate ion prepares the enzyme for attack on the incoming substrate. The mutated TCQH enzyme CysSer, which was used for the studies by Kiefer and Copley,25 is similar in active site composition to MPI, but shows much reduced catalysis, as does the CysAla mutant discussed by Board et al.20 The main difference in the catalytically competent MPI is the presence of His104 in place of the Gln, which is found in TCHQ-DH and MAAI. The LYSYW N-terminal extension of the motif reported by Copley et al. can be replaced in this bacterial group by LYXØØ where ØØ represents a pair of aromatic residues, most frequently YF/W.

isomerases, especially the large number of isomerases observed in bacterial genomes in similar catabolic pathways. The important features appear to be: a means of stabilising the glutathione thiolate to promote substrate attack, a positively charged residue (Arg176 in MPI, Arg175 in MAAI) or base (as observed in TCHQ) to promote the direct binding or formation in the active site of the enone form of the substrate, and the positioning of side chains, which provide sufficient interactions for the substrate to bind, but better complementarity to the product, to promote product release. In addition, analysis of the N200 sequences available for similar enzymes suggests that the Zeta family has at least two distinct subgroups, with alternative features in the active site. The first eukaryotic enzymes have the motif RSSCxWRVRIAL (first identified by Board et al.3) with a serine and a cysteine in the active site, partnered by a glutamine at the N terminus of helix α4 that stabilises the glutathione thiolate, and extended by Anandarajah et al. to LYSYWR/LSSCSXR/KVRIAL.13 We have also identified a group of N 140 prokaryotic sequences with the consensus LYXYF/WRSSASYRVRIAL, represented by MPI, which have no cysteine in the motif and therefore in the active site of the enzyme, and which also all have a histidine in the position where glutamine is observed in the first subgroup. The MPI structure suggests that the thiolate is stabilised by this histidine at the N terminus of helix α4 in this group of enzymes.

Conclusions

Recombinant nagL was expressed in E. coli Rosetta strain in the pET28a vector (Novagen) with an N-terminal His tag. The enzyme was purified in a single step by affinity chromatography on Ni2+ column (Novagen). Protein eluted at 15 mg/ml and was dialysed against crystallisation buffer containing 20 mM Tris (pH 7.4), 2 mM glutathione, 1 mM DTT and 20 mM imidazole. Plateshaped crystals were obtained at 18 °C by the hanging-

The structure of the MPI–DCEG complex presented here gives possible insight into the binding and isomerisation of the substrate maleyl pyruvate. The observations and predictions about the mechanism of isomerisation are potentially applicable to other

Materials and Methods Expression, purification and crystallisation

Maleyl Pyruvate Isomerase: Mechanism of a ζ-GST

175

Table 1. Data collection and refinement statistics

A. Data collection Space group Unit cell dimensions a (Å) b (Å) c (Å) α = γ (°) β (°) Resolution range (Å) (Outer shell) Unique reflections (Redundancy) Rsym % Completeness (Outer Shell) % Mean I/σI B. Refinement Data range (all data with F N 0.0) No. reflections in work set (test set) R-factor (RfreeB); (Outer shell) Rms dev. from ideality: bonds (Å); angles bB-factorN

drop, vapour-diffusion method with wells containing 20% PEG 10,000 in 0.1 M Hepes buffer (pH 7.5). The resulting plate-like crystals belong to the space group P212121 with cell dimensions a = 60.3 Å, b = 81.7 Å, c = 88.2 Å, and diffract anisotropically. Dialysis of apo MPI against the crystallisation buffer resulted in the protein precipitating. To obtain a structure with DCEG, the protein was incubated with 2 mM DCEG directly after affinity purification. The protein was then subjected to buffer exchange into the crystallisation buffer (20 mM Tris pH 7.4, 1 mM DTT, 20 mM imidazole) using a desalting column. Diamondshaped crystals (P21, a = 52.6 Å, b = 60.3 Å, c = 81.7 Å, α = γ = 90.0°, β = 93.2°) were obtained in hanging drops using Clear Strategy screen 1 (Molecular Dimensions Ltd). The drops were composed of 1 μl of protein in mother liquor (20 mM Tris (pH 7.4), 2 mM DCEG, 1 mM DTT and 20 mM imidazole), 1 μl of reservoir solution (100 mM Tris (pH7.5) in 0.3 M sodium acetate, 8% PEG 20,000 and 8% PEG 550 MME). Structure solution and refinement For the holo structure, diffraction data, 97.3% complete to 2.3 Å resolution, were collected at 100K using synchrotron radiation at station 14.2 at the Daresbury Laboratory SRS and reduced using HKL-2000 (HKL Industries, plc) (Table 1). The structure was solved by molecular replacement with BEAST26 using the protein coordinates of human maleyl acetotactetate isomerase (1FW1).10 Phase improvement taking advantage of the twofold NCS was performed using Resolve.27 The starting model was initially refined using simulated annealing in CNS.28 The structure was built using O,29 refined in REFMAC with 5% of the data set aside as a free set, and solvent sites were identified using programmes in the CCP4 suite.30 The final model comprises 3680 atoms in two protein chains, two molecules of glutathione and 302 water molecules, and has an RcrystB = 22.1% (highest resolution shell 27.6%) (RfreeB = 27.5% (highest resolution shell 36.6%)). Overall, the model shows good stereochemistry. All non-glycine and non-proline residues lie within the allowed regions of the Ramachandran plot, with 94.3% lying in the most favoured regions and 5.7% in the additionally allowed regions. The root-mean-square deviation (RMSD) for bond lengths was 0.016 Å and 1.6° for bond angles.

Holo complex

DCEG

P212121

P21

60.3 81.7 88.2 90 90 30.0–2.3 19,625 (4.3) 12.3 (33.2) 97.3 (93.8)

52.6 60.3 81.7 90 93.2 27.6 – 1.2 (1.27–1.2) 394,142 (3.3) 3.4 (39.6) 74.7 (11.9) 16.8 (2.3)

30.0 – 2.3 Å 18,584 (950) 21.5 (26.7); 28.0 (34.9) 0.016; 1.58 21.0

27.0 – 1.3 106,788 (5652) 15.0 (16.5);20.3 (23.3) 0.006; 1.19 11.9

Diffraction data for the complex, which diffracted strongly even at 1.2 Å resolution, were collected at BM14, ESRF Grenoble and reduced using the CCP4 suite. The data are 99% complete to 1.5 Å. The deposited structure was refined against data up to 1.3 Å, which was 44% complete in the outer shell. The final model comprises 3680 atoms in two protein chains, two molecules of glutathione and 302 water molecules and has an RcrystB = 15.0% (highest resolution shell 20.7%) (RfreeB = 16.5% (highest resolution shell 23.3%)). Model building and structure analysis Models of maleyl pyruvate, an intermediate with a single bond in the C2–C3 position, and fumaryl pyruvate were built in Coot,31 and manipulated using dictionaries generated in Sketcher in the CCP4 package. The protein structure in the MPI–DCEG complex was chosen as a basis for the model building. The crystal structures of human,10 mouse (2CZ2 and 2CZ3, unpublished results) and Arabidopsis thaliana12 maleyl acetoactetate isomerase were overlaid on MPI for comparison. The overlays were performed using SSM32 in CCP4MG33,34 or Coot.31 The RMSD between the positions of the Cα carbons for these residues was around 0.9 Å2 in each case. Sequence alignment A Blast search35 was performed using the interactive facility at EBI†, with the sequence of Ralstonia MPI as probe (Swiss Ref O86043). The top 200 complete sequences were identified with sequence identity N 40%. These were aligned and the tree calculated using ClustalW,36 as implemented at the EBI. Data bank accession numbers Coordinates and structure factors for the holo structure have been deposited in the RCSB Protein Data Bank with accession codes 2c1r and r2c1rsf. The enzyme collection † http://www.ebi.ac.uk

176 number for MPI is 5.2.1.2. The nucleotide sequence for the gene has been deposited in the EMBLdatabase under accession number AF036940; AAD12621.1. The amino acid sequence of this protein can be accessed through UNIPROT Database under accession number O86043 9RALS. The atomic coordinates and structure factors for the crystal structure of this protein are available in the Protein Structure Database under accession numbers 2jl4 and r2jl4sf (holoenzyme) and 2v6k and r2v6ksf for the MPI–DCEG complex.

Acknowledgements Access to BM14 at the ESRF was funded by the Research Infrastructure Action FP6 program qIntegrating Activity on Synchrotron and Free Electron Laser Scienceq (IA-SFS). We would like to thank the EMBL Grenoble Outstation and in particular, Martin Walsh, for providing support for measurements at the ESRF under the European -Research Infrastructure Action FP6 program.

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