Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site
2003; Springer Nature; Volume: 22; Issue: 11 Linguagem: Inglês
10.1093/emboj/cdg275
ISSN1460-2075
AutoresMichael Arand, B.M. Hallberg, Jin-Yu Zou, Terese Bergfors, Franz Oesch, Mariët J. van der Werf, J.A.M. de Bont, T. Alwyn Jones, Sherry L. Mowbray,
Tópico(s)Mass Spectrometry Techniques and Applications
ResumoArticle1 June 2003free access Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site Michael Arand Michael Arand Department of Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany Search for more papers by this author B.Martin Hallberg B.Martin Hallberg Department of Cell and Molecular Biology, Uppsala University, BMC, Box 596, S-751 24 Uppsala, Sweden Present address: Royal Institute for Technology, Department of Biotechnology, AlbaNova University Centre, 106 91 Stockholm, Sweden Search for more papers by this author Jinyu Zou Jinyu Zou Department of Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany Search for more papers by this author Terese Bergfors Terese Bergfors Department of Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany Search for more papers by this author Franz Oesch Franz Oesch Institute of Toxicology, University of Mainz, Obere Zahlbacherstrasse 67, D-55131 Mainz, Germany Search for more papers by this author Mariët J. van der Werf Mariët J. van der Werf Division of Industrial Microbiology, Wageningen Agricultural University, 6700 EV Wageningen, The Netherlands Present address: Department of Applied Microbiology and Gene Technology, TNO Voeding, PO Box 360, 3700 AJ Zeist, The Netherlands Search for more papers by this author Jan A.M. de Bont Jan A.M. de Bont Division of Industrial Microbiology, Wageningen Agricultural University, 6700 EV Wageningen, The Netherlands Present address: TNO Environment, Energy and Process Innovation, PO Box 342, 7300 AH Apeldoorn, The Netherlands Search for more papers by this author T.Alwyn Jones T.Alwyn Jones Department of Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany Search for more papers by this author Sherry L. Mowbray Corresponding Author Sherry L. Mowbray Department of Molecular Biosciences, Swedish University of Agricultural Sciences, BMC, Box 590, S-751 24 Uppsala, Sweden Search for more papers by this author Michael Arand Michael Arand Department of Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany Search for more papers by this author B.Martin Hallberg B.Martin Hallberg Department of Cell and Molecular Biology, Uppsala University, BMC, Box 596, S-751 24 Uppsala, Sweden Present address: Royal Institute for Technology, Department of Biotechnology, AlbaNova University Centre, 106 91 Stockholm, Sweden Search for more papers by this author Jinyu Zou Jinyu Zou Department of Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany Search for more papers by this author Terese Bergfors Terese Bergfors Department of Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany Search for more papers by this author Franz Oesch Franz Oesch Institute of Toxicology, University of Mainz, Obere Zahlbacherstrasse 67, D-55131 Mainz, Germany Search for more papers by this author Mariët J. van der Werf Mariët J. van der Werf Division of Industrial Microbiology, Wageningen Agricultural University, 6700 EV Wageningen, The Netherlands Present address: Department of Applied Microbiology and Gene Technology, TNO Voeding, PO Box 360, 3700 AJ Zeist, The Netherlands Search for more papers by this author Jan A.M. de Bont Jan A.M. de Bont Division of Industrial Microbiology, Wageningen Agricultural University, 6700 EV Wageningen, The Netherlands Present address: TNO Environment, Energy and Process Innovation, PO Box 342, 7300 AH Apeldoorn, The Netherlands Search for more papers by this author T.Alwyn Jones T.Alwyn Jones Department of Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany Search for more papers by this author Sherry L. Mowbray Corresponding Author Sherry L. Mowbray Department of Molecular Biosciences, Swedish University of Agricultural Sciences, BMC, Box 590, S-751 24 Uppsala, Sweden Search for more papers by this author Author Information Michael Arand1, B.Martin Hallberg2,3, Jinyu Zou1, Terese Bergfors1, Franz Oesch4, Mariët J. van der Werf5,6, Jan A.M. de Bont5,7, T.Alwyn Jones1 and Sherry L. Mowbray 8 1Department of Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany 2Department of Cell and Molecular Biology, Uppsala University, BMC, Box 596, S-751 24 Uppsala, Sweden 3Present address: Royal Institute for Technology, Department of Biotechnology, AlbaNova University Centre, 106 91 Stockholm, Sweden 4Institute of Toxicology, University of Mainz, Obere Zahlbacherstrasse 67, D-55131 Mainz, Germany 5Division of Industrial Microbiology, Wageningen Agricultural University, 6700 EV Wageningen, The Netherlands 6Present address: Department of Applied Microbiology and Gene Technology, TNO Voeding, PO Box 360, 3700 AJ Zeist, The Netherlands 7Present address: TNO Environment, Energy and Process Innovation, PO Box 342, 7300 AH Apeldoorn, The Netherlands 8Department of Molecular Biosciences, Swedish University of Agricultural Sciences, BMC, Box 590, S-751 24 Uppsala, Sweden *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:2583-2592https://doi.org/10.1093/emboj/cdg275 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Epoxide hydrolases are essential for the processing of epoxide-containing compounds in detoxification or metabolism. The classic epoxide hydrolases have an α/β hydrolase fold and act via a two-step reaction mechanism including an enzyme–substrate intermediate. We report here the structure of the limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis, solved using single-wavelength anomalous dispersion from a selenomethionine-substituted protein and refined at 1.2 Å resolution. This enzyme represents a completely different structure and a novel one-step mechanism. The fold features a highly curved six-stranded mixed β-sheet, with four α-helices packed onto it to create a deep pocket. Although most residues lining this pocket are hydrophobic, a cluster of polar groups, including an Asp–Arg–Asp triad, interact at its deepest point. Site-directed mutagenesis supports the conclusion that this is the active site. Further, a 1.7 Å resolution structure shows the inhibitor valpromide bound at this position, with its polar atoms interacting directly with the residues of the triad. We suggest that several bacterial proteins of currently unknown function will share this structure and, in some cases, catalytic properties. Introduction Epoxide hydrolases (EHs) catalyse the hydrolysis of epoxides to the corresponding diols. In nature, these enzymes have three main functional roles: detoxification, synthesis of signal molecules, and metabolism which allows some bacteria to use epoxides or their alkene and halohydrin precursors as carbon sources. EHs have also gained increasing attention due to their potential for kinetically resolving epoxide enantiomers and thus serving as selective catalysts in the industrial production of enantiomerically pure chemicals. The most extensively investigated group of EHs can convert highly reactive, and therefore often harmful, epoxides into less toxic and more easily excreted diols; these enzymes usually act with rather broad substrate specificity (Armstrong, 1987). The structures of three related vicinal diol-producing EHs from bacterial, mammalian and fungal sources have been solved to date (Argiriadi et al., 1999; Nardini et al., 1999; Zou et al., 2000). All have a characteristic α/β hydrolase fold (Heikinheimo et al., 1999), combined with a helical lid domain. The catalytic triad of this family is situated on the α/β domain, and consists of an aspartic acid nucleophile and a histidine–acidic-residue pair that acts as a charge relay (Franken et al., 1991). Two tyrosines from the lid help to position the substrate and encourage ring opening through hydrogen bonds to the epoxide oxygen (Argiriadi et al., 2000; Rink et al., 2000; Yamada et al., 2000). The nucleophile attacks primarily at the less-substituted carbon of the oxirane ring to form an enzyme–substrate intermediate. The intermediate is then attacked by a water molecule activated by the charge relay pair. The structure of human leukotriene A4 hydrolase, a bifunctional enzyme with epoxide hydrolase activity, has recently been solved (Thunnissen et al., 2001). This metalloenzyme catalyses the biosynthesis of leukotriene B4, a potent lipid chemoattractant involved in inflammatory and other responses. The catalytic unit has a fold similar to thermolysin, i.e. two domains with structures very different from those of the α/β hydrolase EHs. The enzyme has an atypical EH activity, in the sense that its product is not a vicinal diol; the hydroxyl group introduced is distant from the oxirane ring. The zinc atom in the active site is proposed to activate the epoxide to form a carbocation intermediate that is delocalized through a conjugated system, thus enabling an activated water molecule to attack at the opposite end of that system. The subject of the present study, limonene-1,2-epoxide hydrolase (LEH) (EC 3.3.2.8), catalyses the reaction shown in Figure 1. This 16 kDa protein is part of a metabolic system that allows the bacterium Rhodococcus erythropolis to grow on limonene as a sole source of carbon and energy (van der Werf et al., 1999c). Since the enzyme is much smaller than, and lacks sequence similarity to, the other types of epoxide hydrolases, it was immediately suspected that it would have a different fold (Barbirato et al., 1998; van der Werf et al., 1998). Furthermore, LEH has been shown to use a different (acid-catalysed) mechanism with different stereochemical consequences (van der Werf et al., 1999a). No detectable enzyme–substrate intermediate was found in studies where enzyme was incubated with 14C-labelled substrate (M.Arand, unpublished results). In addition to its structural and mechanistic interest, LEH has many potential applications in industrial synthesis, particularly since its reaction is enantioconvergent. For example, the (1S, 2R, 4S) and (1R, 2S, 4S) isomers of limonene epoxide are both converted to the (1R, 2R, 4S) diol, while the (1S, 2R, 4R) and (1R, 2S, 4R) isomers are each converted to the (1S, 2S, 4R) diol (van der Werf et al., 1999b). These results indicate that a water molecule must be able to attack at either C1 or C2 of the epoxide ring (van der Werf et al., 1999a). Figure 1.The reaction of LEH with its natural substrate. Carbon atoms are numbered. Download figure Download PowerPoint We report here the structure of LEH from R.erythropolis, the first representative of this third type of epoxide hydrolase, at 1.2 Å resolution. It has a similar topology to several other enzymes of diverse function but very different reaction mechanisms. As for all but one of these enzymes, the active site of LEH lies at the bottom of a deep pocket that is lined primarily with hydrophobic residues. The catalytic importance of several polar residues found at the bottom of the pocket was confirmed by site-directed mutational studies, as well as by a 1.75 Å crystal structure of a complex with the inhibitor valpromide. The combined results suggest a one-step push–pull reaction mechanism. Further, we were able to suggest a function for four putative EHs that show sequence similarity to LEH, including conserved catalytic residues. So far these related proteins have been found only in pathogens, and may represent part of their defence against foreign compounds. Results Overall structure The structure of LEH was solved by single-wavelength anomalous dispersion (Hendrickson and Teeter, 1981) using a selenomethionine-substituted (Se-Met) protein expressed in Escherichia coli. Statistics describing the X-ray diffraction data and the final refined native LEH model are provided in Tables I and II. There is no electron density for the first four residues of either molecule in the asymmetric unit. Of the remainder, only residues 14–17 of one molecule (molecule A in our PDB deposition) have relatively poor electron density. Table 1. Data collection statistics Native LEH Se-Met LEH Valpromide Unit cell dimensions (Å) a = 45.6 a = 45.4 a = 45.6 b = 47.6 b = 47.6 b = 47.7 c = 129.7 c = 129.3 c = 129.2 Wavelength (Å) 0.931 0.934 0.944 Resolution range (Å) 64.6–1.2 (1.20–1.21) 38–1.8 (1.8–1.9) 30.0–1.75 (1.75–1.80) No. of unique reflections 88 916 26 770 29 009 Multiplicity 4.1 (2.3) 7.0 (7.1) 3.9 (3.6) Completeness (%) 99.7 (94.1) 100 (100) 99.3 (97.5) Rsym (%)a 6.3 (27.2) 6.6 (32.0) 5.6 (10.7) Average I/σ(I)b 28.2 (3.5) 8.8 (2.3) 20.7 (12.2) Figure of meritc – 0.20 (0.09) – Cullis R-factor, anomalousc,d – 0.83 (0.98) – Figure of merite – 0.44 (0.15) – All forms of LEH crystallized in space group P212121. Values in parentheses refer to data in the outer resolution shell. a Rsym = ∑∑I|II − |/∑ , where is the mean intensity of the N reflections with intensities II and common indices h,k,l. b As reported by TRUNCATE (French and Wilson, 1978). c As reported by MLPHARE (Otwinowski, 1991). d Cullis R-factor = (|FPHi(+) − FPHi(−)| − 2FHi × sin(PHIx)/|FPHi(+) − FPHi(−)|, where PHIx is the protein phase. e Output by RESOLVE (Terwilliger, 2000). Table 2. Statistics for the final refined model Model 'apo' Valpromide Resolution range (Å) 50–1.2 29–1.75 No. of reflections 84 491 27 485 No. of reflections used for Rfree calculation 4306 1469 R value, Rfree value (%) 14.5, 17.2 15.8, 19.5 No. of non-hydrogen atoms 2712 2643 No. of riding hydrogen atoms 3073 2932 No. of solvent waters 389 330 Mean B-factor, protein (Å2)a 15.5 14.7 Average B-factor, solvent atoms (Å2)a 29.1 26.0 Average B-factor, ligand atoms (Å2) 24.3 25.7 Ramachandran plot outliers (%)b 0 0 R.m.s.d. bond length from ideal (Å)c 0.025 0.018 R.m.s.d. bond angle from ideal (deg)c 2.0 1.9 R.m.s.d., root mean square deviation. a Protein atoms were modelled using anisotropic temperature factors, and solvents were modelled with isotropic temperature factors. b Using a stringent boundary Ramachandran plot (Kleywegt and Jones, 1996). c Ideal values as defined by Engh and Huber, 1991. The main fold consists of a six-stranded mixed β-sheet, with three α-helices packed on to one side (Figure 2). Three of the strands (β3–β5) are long and highly curved. In β3, the curvature is achieved by bulges at residues 81 and 86, while in β4 and β5 it is the result of gradual changes in the (φ, ψ) angles of successive residues. The packing of the three N-terminal helices onto the concave face of the sheet creates a pocket that extends ∼15 Å into the protein core. A fourth helix lies beside the long strands of the sheet in such a way that it acts as a structural extension of the three shorter strands and a rim to the pocket. The residues lining this pocket are mainly hydrophobic, with the exception of a cluster of polar and charged residues at its deepest point (Figure 3). Figure 2.Structure of LEH. (A) Ribbon drawing showing the dimer, with each subunit coloured going through the rainbow from red at the N-terminus to blue at the C-terminus. Some of the residues contributing to the dimer interface, as described in the text, are shown as ball-and-stick representations. The endogenous ligand is also shown (magenta) in both subunits. (B) Topology diagram of the LEH subunit, using the same rainbow scheme. Residues included in each secondary structural element are numbered; helix α2 is irregular. Active-site residues are indicated by magenta stars. Download figure Download PowerPoint Figure 3.The active site. (A) Catalytic residues, showing their relationship to each other and supporting side-chains, as well as to the water molecule and endogenous ligand found in the LEH active site. Hydrogen-bonding interactions are shown by dotted lines. Colouring of the ribbon portions follows the rainbow scheme defined in Figure 2. The electron density of the endogenous ligand (modelled as heptanamide) in the final 2Fo − Fco map is contoured at a level of 1σ. (B) Hydrogen-bonding interactions between active-site groups and the endogenous ligand. The figure shows Asp132 acting as the base and Asp101 as the acid. Where the donor–acceptor relationship is not clear from the available data, hydrogen bonds are indicated by double-headed arrows. (C) Complex with valpromide. The electron density of the final 2Fo − Fco map is contoured at a level of 1σ. Download figure Download PowerPoint As predicted (Barbirato et al., 1998; van der Werf et al., 1998), the LEH structure does not resemble those of the epoxide hydrolases described previously. However, the folding topology is not new; a search of the Protein Data Bank (PDB) (Bernstein et al., 1977; Berman et al., 2000) identified four similar proteins (Table III). The most closely related structures are 3-oxo-Δ5-steroid isomerase (Kim et al., 1997; Cho et al., 1998) and a nuclear transport factor NTF2 (Bullock et al., 1996). Those of scytalone dehydratase (Lundqvist et al., 1994) and the β subunit of an indoaromatic-ring-hydroxylating naphthalene 1,2-dioxygenase (Kauppi et al., 1998) are more distantly related. Each protein exhibits the distinctive pocket formed when the helices pack onto the β-sheet. All show insignificant sequence conservation to LEH, and could only be identified with the structure in hand. Table 3. Structural alignments to LEH PDB code Name No. of atoms matching Aggregation state RMS distance % ID of matched At Tyr53 At Asn55 At Arg99 At Asp101 At Asp132 1OPY 3-oxo-Δ5-steroid isomerase (Pseudomonas putida) 107/123 Dimer 1.54 17.8 Val38 Asp40 Phe86 Val88 Ala118 1ASK NTF2 99/125 Dimer 1.35 8.2 Leu39 Trp41 Gly87 Leu89 Phe119 4STD Scytalone dehydratase 88/164 Trimer 1.75 9.1 Ile48 Tyr50 Leu106 Val108 Pro149 1NDO Naphthalene 1,2-dioxygenase 86/143 α3β3 hexamer 1.66 5.9 Tyr564 Val566 Val641 Leu643 Val678 Although LEH was previously reported to be a monomer on the basis of gel filtration experiments (van der Werf et al., 1998), it appears as a dimer in the asymmetric unit of the crystal (Figure 2A). The subunits are related by 179°, corresponding to a nearly perfect 2-fold axis. Only the residues at the N-terminus (i.e. those preceding the main fold) deviate significantly from the dimer symmetry. After least-squares alignment, the Cα atoms of residues 20–149 match with an RMS distance of 0.2 Å; the RMS difference of their independently refined temperature factors is 1.1 Å2. The β-sheets of the two subunits are packed perpendicular to each other, burying a total solvent accessible area of 3100 Å2. The surfaces have a highly complementary shape and a large number of side-chain–side-chain interactions are formed. The observed temperature factors for atoms in this interface are very similar to those found in the core of the protein, and significantly lower than those of surface residues. The Leu117 side-chains are completely buried at the centre, each making direct van der Waals contact with its equivalent, as well as with Tyr133 of the opposing subunit. A number of interactions result from the juxtaposition of the N-terminal ends of the α4s. The Asp135 side-chains hydrogen bond to the main-chain nitrogens of residues 136. Each Arg137 stacks onto Arg148 of the other subunit, and the arginines simultaneously make bidentate salt-link interactions with Glu140 and Glu141, respectively, across the interface. The two Tyr96 residues stack onto each other, while guanidino-aromatic stacking interactions are formed by Arg9 and Tyr62, as well as Arg131 and Trp10; Arg131 simultaneously forms a salt link with Glu98 across the dyad axis. Several water molecules are also found as part of the interface, some of which obey the 2-fold symmetry. No main-chain–main-chain hydrogen bonds are observed between the two subunits. Active site The residues responsible for LEH catalysis had not been identified in any previous study. Our inspection of the proteins with similar structure indicated that, in all but one case, the active site is found in the deep pocket between the β-sheet and the three N-terminal α-helices. The exception is the β subunit of naphthalene 1,2-dioxygenase, where the C-terminus of the polypeptide chain is inserted into the pocket and the active site is situated elsewhere (Carredano et al., 2000). These structurally related proteins have widely diverse functions, and the residues lining the pockets are not conserved; as a result they do not offer further guidance for the analysis of the LEH mechanism. Although most of the side-chains lining the LEH pocket are hydrophobic, a cluster of polar residues is found at its deepest point where the side-chain of Arg99 interacts with the carboxylate groups of Asp101 and Asp132 (Figure 3). In the experimentally phased map for the Se-Met-substituted enzyme, electron density for an unexpected ligand was observed to stretch from these polar residues to the surface of the protein (Figure 3A). Clear density persisted during the high-resolution refinement of the native protein. The hydrogen-bonding interactions linking the various groups are summarized in Figure 3B. A short distance between the side-chain of Asp101 and the observed ligand (2.5 Å) indicates a strong hydrogen-bonding interaction. A second short hydrogen bond (2.6 Å) links the ligand and a well-defined water molecule, which in turn interacts with the side-chains of Tyr53, Asn55 and Asp132. All these side-chains are contributed by the β-sheet (Figure 2B). In the final stages of the refinement, the ligand was modelled as heptanamide, which is an excellent match for the shape of the electron density and the chemical character of this pocket. However, we have not yet been able to identify the actual compound experimentally. The native and Se-Met proteins were expressed in different hosts and purified using distinct procedures, yet both show clear density for this ligand. The precipitant in the crystallization represents the sole common experimental parameter, and so it seems likely that the ligand is an undocumented impurity of the polyethylene glycol (PEG) 6000 used. The electron density is not consistent with PEG itself. With an added concentration of 30% (w/v) PEG, the impurity need not be present in large quantities if its binding constant is sufficiently favourable. For example, if the Kd were 100 μM, a 0.03% impurity would be adequate for 90% occupancy of the site under our crystallization conditions. Inhibition studies with high concentrations of PEG 6000 were precluded for technical reasons, but PEG 1500 (which might be expected to have similar impurities) does indeed inhibit, with an effective Ki of 7% (w/v). We have not yet been able to obtain diffracting crystals without PEG. Based on the above information, we tested the inhibition of LEH activity with four commercially available compounds expected to have chemical characteristics similar to the endogenous ligand. Hexanamide, hexylamine and valpromide (dipropylacetamide) were found to act as competitive inhibitors of LEH (Ki values of 2 mM, 35 μM and 100 μM, respectively). Hexanoic acid did not inhibit significantly at a concentration of 1 mM, which is probably not surprising given the expected repulsion between its negatively charged carboxylate group and those of the active site. A complex of LEH with valpromide was obtained through crystal soaking experiments and refined at 1.75 Å resolution. This inhibitor bound in the same manner in the two subunits (Figure 3C), with clear electron density for its amide group appearing in an identical location to the putative amide group of the endogenous ligand (compare Figure 3A). A water molecule is again firmly positioned by hydrogen bonds to the side-chains of Tyr53, Asn55 and Asp132. In both active sites, the strongest electron density was for the propyl group that follows the path of the fatty acid tail of the endogenous ligand. The side-chain conformation of Leu74 was altered, apparently to accommodate the second propyl group of valpromide. The conformation of Leu103 was also different, and a similar conformational change in Leu147 is accompanied by a more general movement of the C-terminal residues 145–149 in both subunits. The side-chain conformation of Phe139 is changed slightly in both active sites. When combined, these changes illustrate how the hydrophobic lining of the active-site pocket of LEH can be adapted to fit differently shaped substrates. Site-directed mutations The structural and functional importance of the Asp–Arg–Asp triad and nearby residues was further evaluated using site-directed mutations. In total, 12 mutants were constructed, targeting the five polar residues that could reasonably have direct roles in catalysis, namely Tyr53, Asn55, Arg99, Asp101 and Asp132 (Table IV). Some of the mutant proteins formed inclusion bodies when expressed at 30°C, but growth of the bacteria at 26°C allowed the production of apparently soluble mutant protein in each case. However, folding was aberrant for some mutant proteins, as judged by their behaviour during ion exchange chromatography where they eluted at much higher salt concentrations than the wild-type LEH (Table IV). For some mutants, improper folding was further suggested by a slow-growth phenotype and somewhat reduced expression (Table IV). Mutations at Arg99 were particularly prone to these types of behaviour. Table 4. Comparison of wild-type and mutant LEH enzymes Residue Replaced with Growth Expression level (% of total protein) [NaCl] (mM) needed to elute from ion-exchange column kcat (s−1) Km (mM) kcat/Km (s−1M−1) WT – Normal 20–40 210 0.470 1.4 340 Tyr53 Phe Normal 20–40 210 0.170 3.7 46 Asn55 Ala Normal 20–40 215 0.002 1.4 1.64 Asn55 Asp Normal 20–40 580 ND ND ND Arg99 Ala Slow 3–10 560 ND ND ND Arg99 Lys Slow 3–10 530 ND ND ND Arg99 Gln Slow 3–10 500 ND ND ND Arg99 His Slow 3–10 545 ND ND ND Asp101 Ala Normal 20–40 170 ND ND ND Asp101 Asn Normal 20–40 215 ND ND ND Asp132 Ala Normal 20–40 455 ND ND ND Asp132 Asn Normal 20–40 195 ND ND ND Asn55/Asp132 Asp/Asn Normal 20–40 240/480a ND ND ND Mutants that showed any measurable activity in an initial assay with 60 μM of racemic styrene-7,8-oxide were characterized further. ND, not detectable. The threshold of detectable activity was ∼0.1% of that of the wild-type enzyme. a Two peaks were observed for this mutant. The purified recombinant wild-type enzyme converts racemic styrene-7,8-oxide with a turnover number of 0.47 s−1. Although this substrate is somewhat poorer than the natural limonene-1,2-epoxide, which has a turnover number of 23.4 s−1 (van der Werf et al., 1998), use of styrene oxide allowed us to perform a partition assay that is faster, easier and much more sensitive than the traditional gas chromatographic measurement of limonene epoxide hydrolysis. The mutant activity results, summarized in Table IV, are in complete agreement with the proposal that the central pocket of LEH contains its active site. The members of the Asp101–Arg99–Asp132 triad are clearly most important. Both the aspartic acid residues are essential for enzyme activity. Although all mutations of Arg99 were also catalytically inactive, the structural roles of this residue mean that its contribution to catalysis cannot be viewed in isolation. However, its intimate associations with Asp101 and Asp132 (four hydrogen bonds) strongly imply that it will act together with them. The residues that position the observed water molecule are also important, although less central to activity. Replacement of Tyr53 with phenylalanine resulted in a partially active mutant. Mutation of Asn55 to the isosteric aspartic acid generated an inactive enzyme, probably due in part to a local negative charge overload; the biochemical data suggest that some of this mutant protein may be misfolded (Table IV). A double mutation Asn55Asp/Asp132Asn that restores the local charge rescues folding, but not activity, and again illustrates the importance of Asp132 in catalysis. Mutation of Asn55 to the smaller side-chain of alanine left a small residual activity, possibly because of non-optimal positioning of the catalytic water. A similar explanation may lie behind the partial activity of the Tyr53Phe mutant. In summary, the mutagenesis data, like the structural observations, are consistent with the proposal that the water molecule associated with Asp132, Asn55 and Tyr53 is the hydrolytic water and Asp101 is the acid catalyst that protonates the epoxide oxygen. Comparison with other proteins of unknown structure Searches of the sequence databases identified several hypothetical proteins with sequence similarity to LEH (Figure 4). The sequences fall into two groups. Figure 4.Sequence alignments of LEH with proteins of unknown function. Sequence alignment was performed using hidden Markov models (Karplus et al., 1998), with some small manual adjustments using the LEH structure as a guide. Every tenth residue in the LEH sequence is marked
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