Enantioselectivity of recombinant Rhizomucor miehei lipase in the ring opening of oxazolin-5(4H)-ones

Nigel A. Turner1, Duncan J. H. Gaskin1, Asutosh T. Yagnik2,3, Jennifer A. Littlechild2 and Evgeny N. Vulfson1,4

1 Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, and 2 Schools of Chemistry and Biological Sciences, Exeter University, Exeter EX4 4QD, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
Enantioselectivity of enzyme catalysis is often rationalized via active site models. These models are constructed on the basis of comparing the enantiomeric excess of product observed in a series of reactions which are conducted with a range of homologous substrates, typically carrying various side chain substitutions. Surprisingly the practical application of these simple but informative `pocket size' models has been rarely tested in genetic engineering experiments. In this paper we report the construction, purification and enantioselectivity of two recombinant Rhizomucor miehei lipases which were designed to check the validity of such a model in reactions of ring opening of oxazolin-5(4H)-ones.

Keywords: biotransformation/molecular modelling/protein engineering/recombinant lipase/transesterification


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
The selectivity of enzymes is one of the features that makes them so attractive for many industrial applications. In particular, the use of esterases for the resolution of racemic mixtures both on industrial and laboratory scales has become a common practice in recent years (for review see Chen and Sih, 1989; Klibanov, 1989; Faber and Franssen, 1993; Santaniello et al., 1993; Theil, 1995; Kazlauskas and Bornscheuer, 1998). The commercial availability of numerous esterases has made it relatively easy to select a suitable biocatalyst to perform a required resolution. However, the transfer of resolution protocols from laboratory to industrial scale still remains a challenging problem. Indeed, not too many esterase/lipase preparations are sufficiently inexpensive and robust to be used in large scale processing (Bjorkling et al., 1991Go; Andersson et al., 1998Go). The process economics not only demands high enantioselectivity but also high operational stability of the bioctalyst to satisfy the requirements of modern industrial operations. Thus, it is more attractive, from a practical standpoint, to attempt `fine tuning' of the enantioselectivity of those enzymes which are known to be suitable for large scale applications (e.g. cheap, highly active, thermostable and solvent resistant), than to embark on a search for a new enzyme with all these properties as well as the desired stereoselectivity.

There is a very extensive literature on the use of lipases for the resolution of racemates (see references above) and in numerous investigations a wide range of homologous substrates have been tested to probe enzyme selectivity and rationalize it in terms of substrate binding within the active site (Toone et al., 1990Go; Burgess and Jennings, 1991Go; Kazlauskas et al., 1991Go; Tanaka et al., 1993Go; Faber et al., 1994Go; Grochulski et al., 1994Go; Provencher and Jones, 1994Go; Holmquist et al., 1996Go; Lemke et al., 1997Go; Naemura et al., 1995Go, 1996Go). These simple but informative chemical models often suggest that it is steric constraints, associated with the binding of one enantiomer as compared to the other in the active site `pocket', which are essentially responsible for the selectivity observed. Similarly, molecular modelling studies investigating such lipase reactions have suggested several possible reasons for the observed selectivities (Kazlauskas 1994; Lawson et al., 1994Go; Norin et al., 1994Go; Uppenberg et al, 1995Go; Jaaskelainen et al., 1996Go; Carriere et al., 1998Go; Haeffner et al., 1998Go; Scheib et al., 1998Go). It is somewhat surprising, however, that with a few notable exceptions (see, for example, Kazlauskas, 1994; Scheib et al., 1998) these models have not been verified by genetic engineering as in many cases they offer a clear prediction of the effect of the size of the active site pocket on the enzyme's enantioselectivity. This paper sets out to check experimentally the validity of such an approach.

Rhizomucor miehei lipase (RML; E.C. 3.1.1.3) was chosen for the study as this enzyme has been used successfully in several industrial processes (Bjorkling et al., 1991Go; Vulfson, 1994Go) and its high resolution 3-D structure has been determined (Brady et al., 1990Go; Brzozowski et al., 1991Go; Derewenda et al., 1992Go). The reaction of ring opening of the oxazolin-5(4H)-ones (1) yielding the esters 2 (Figure 1Go) (Turner et al., 1995Go; Winterman, 1996Go) was selected as a model biotransformation for two reasons. Firstly, the substrates 1a and 1b were esterified using n-butanol and RML with almost absolute (99.5% ee) and virtually no enantioselectivity respectively (Turner et al., 1995Go; Winterman, 1996Go). This provided two homologous substrates/reactions for assessing the effect of site-directed mutagenesis. Secondly, a possible structural basis for the lipase discrimination between these two substrates had been suggested (Yagnik, 1997Go) and from this hypothesis a simple mutagenesis strategy was developed to predict the effect of amino acid substitutions on the enzyme's enantioselectivity. The objective of this work was to assess the validity of such predictions.



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Fig. 1. RML-catalysed ring opening of oxazolin-5(4H)-ones.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
Materials

3-[N-Morpholino]propanesulphonic acid (MOPS), 2-amino-2-(hydroxymethyl)aminomethane (Tris), dithiothreitol (DTT), phenylmethylsulphonyl fluoride (PMSF), tributyrin, Triton-X100, inorganic salts, bovine serum albumin (BSA), benzamidine.HCl, bromocresol purple, Butyl-Sepharose Fast Flow, and UltraGel-HA were obtained from Sigma Chemical Co. (Gillingham, Dorset, UK). KCl, (NH4)2SO4 and EDTA (ethylenediaminetetraacetate) were obtained from Merck (Poole, Dorset, UK). Phenyl-Sepharose and Sephacryl-S200HR were purchased from Pharmacia (Uppsala, Sweden). The electrophoresis materials were purchased from Novex-Electrophoresis (Frankfurt-am-Main, Germany). N-benzoyl-D,L-valine, tert-D,L-leucine, benzoyl chloride, acetic anhydride and all organic solvents were supplied by Aldrich Chemical Co. (Gillingham, Dorset, UK). 1,2-O-Dilauryl-rac-glycero-3-glutaric acid, resorufin ester and Thesit were obtained from Boehringer (Mannheim, Germany). All molecular biology reagents were obtained from Promega (Southampton, UK) or InVitrogen (Groningen, The Netherlands) unless otherwise stated. DNA sequencing reagents were obtained from Perkin–Elmer (Warrington, UK).

Computer-aided simulations

General. All the computational simulations were carried out on Silicon Graphics workstations (Silicon Graphics, USA), running the IRIX operating system (versions 5.2 and 5.3). The QUANTA® software (versions 4.1.1 and 96) (Molecular Simulations, USA) was used for the modelling work. The QUANTA® software employed the CHARMm® forcefield (version 22.2 for QUANTA 4.1.1 and v. 23.3 for QUANTA96). The X-ray coordinates for an RML-inhibitor tetrahedral complex, with PDB (Bernstein et al., 1977Go) identifying code 4TGL (Derewenda et al., 1992Go) was used as a basis for modelling studies, retaining all the crystallographic water molecules. Constraints during simulation allowed all residues within 12 Å of Ser 144 free to move and harmonic constraints of 100 kcal/mol on the rest of the structure. A distance-dependent dielectric with a switching function between 14–15 Å was used to represent electronic effects. In all other cases the default software options were used for calculations, unless otherwise stated.

Modelling of the initial tetrahedral intermediate complexes. The RML–oxazolinone substrate complexes were prepared for molecular dynamics (MD) simulation using previously established procedures (Yagnik et al., 1997Go). CHARMm® scripts were used to minimize the initial tetrahedral complexes with standard constraints, except using a constraining harmonic potential of 50 kcal/mol, followed by MD using standard constraints: the system was heated in the range 0–300 K during 1.2 ps, equilibrated at 300 K during 1.2 ps and simulated at 300 K during 10 ps. In each case a time step of 0.001 ps was used, with the SHAKE function employed to constrain only the bonds containing hydrogen atoms. The constraints were removed and the average structure and isotropic fluctuations calculated. The average coordinates from such MD simulations were compared to those from the starting and minimized tetrahedral complex coordinates. Finally, plots were made for the fluctuation in hydrogen-bonding distance between each RML-oxazolinone:O6 complex and residues Ser 82:NH, Leu 145:NH and Tyr 28:OH. The stereochemical quality of the average MD structures was examined using PROCHECK (version 3.3.1) (Laskowski et al., 1993Go). A molecular surface calculation was performed from within the QUANTA software, using all residues within 15 Å of Ser 144 and the default software options, in order to visualize the shape complementarity between the substrate and enzyme.

Computational mutation studies. The average structure from each MD run was used as an input to Ligplot (version 3.2) (Wallace et al., 1995Go). Residue Val 205 was replaced with Ile and also Leu, from the default side-chain rotamer library within QUANTA. A distance-only based criterion was used for placing the new side chains in reasonable starting conformations. The tetrahedral complexes for each mutated enzyme-substrate complex were minimized using CHARMm scripts and standard constraints. The initial and minimized coordinates of the mutant RML-oxazolinone complexes were compared and the r.m.s. deviation calculated.

Genetic engineering

General. All DNA manipulations were carried out according to standard methods (Maniatis et al., 1989) or manufacturers' manuals unless otherwise specified.

Construction of the mutant genes. The V205I and V205L mutant genes were constructed using `megaprimer' PCR mutagenesis (Sarkar et al., 1990) with a degenerate oligonucleotide. 27 pmol of pPICZ12.39 a Pichia pastoris expression plasmid containing the wild type Rhizomucor miehei lipase gene was used a template in the first PCR along with 18 pmol of a flanking primer lip39 (5'-CGCTTCTAGACTTAAGCTAAGTACAGAGGCCTG-3') and 20 pmol of the mutagenic primer (5'-TGAACGAGATATT(A/C)TTCCTCATCTTC-3'). The product from this reaction was cleaned using a Qiagen PCR clean-up kit (Qiagen Ltd, UK) and used as primer in a second PCR with 5 pmol pPICZ12.39 as template, and 14 pmol of a second flanking primer lip12 (5'-CCGGAATTCGCCATGGCATCGATTGATG-3'). The product from this reaction was cleaned using a Qiagen PCR clean-up kit and cut with EcoRI and XbaI. This fragment was ligated into EcoRI/XbaI cut pPICZ12.39 from which the lipase gene had been removed. The ligation reaction was transformed into Escherichia coli strain TOPF', and plated out. Several colonies were picked, grown up and the DNA extracted. The sequence of individual clones was determined and mutant sequences identified.

Transformation of Pichia pastoris. Pichia pastoris strain GS115 was transformed with 5 µg of DNA from the two mutants according to InVitrogen's protocols. Several zeocin-resistant colonies from each transformation were grown up in 5 ml YPD O/N at 30°C. Genomic DNA was prepared and sequenced in order to confirm the presence of each mutation.

Enzyme purification

For the purification of the recombinant lipases, Pichia pastoris expressing either V205L or V205I was grown in a conical flask as described in detail elsewhere. The fermentation broth (1 l) was clarified by centrifugation at 3000 g for 20 min. To the resulting supernatants (1 l) benzamidine.HCL, DTT, EDTA and PMSF were added to a final concentration of 5, 1, 1, and 0.1 mM, respectively, and the supernatants were dialysed overnight against 2 x 5 l of 10 mM MOPS buffer pH 6.8, containing benzamidine (5 mM), EDTA (1 mM), DTT (1 mM) and PMSF (0.1 mM).

Chromatography on phenyl-Sepharose. Solid ammonium sulphate was added to the dialysed supernatants whilst stirring, up to a final concentration of 0.5 M and this protein solution was applied to a phenyl-Sepharose column (5 x 35 cm) pre-equilibrated with 10 mM MOPS pH 6.8, containing 1 mM EDTA and 0.5 M ammonium sulphate. Once all the protein had been loaded, the column was washed with more of the same buffer (1 l) to elute the non-binding material. The enzyme was eluted by washing the column with distilled water (600 ml).

Chromatography on butyl-Sepharose. The lipase containing fraction from the phenyl-Sepharose column was concentrated by ultrafiltration (Amicon stirred cell, YM-10 membrane) to a volume of ~25 mL and had solid ammonium sulphate added to it to a final concentration of 0.6 M. The enzyme was applied to a butyl-Sepharose FF column (2.5 x 15 cm) equilibrated in 10 mM MOPS pH 6.8, containing 1 mM EDTA and 0.6 M ammonium sulphate. The column was washed with 2 bed volumes of the MOPS/ammonium sulphate buffer to elute non-binding material and then a linear gradient of the MOPS/ammonium sulphate buffer and distilled water was applied. Fractions (6 ml) were collected and assayed for lipase activity using resorufin and tributyrin/bromocresol purple assays (see below). The active fractions were combined, concentrated to a volume of ~10 ml, as described above, and dialysed against distilled water (2 x 1 l).

Chromatography on Ultragel-HA. The enzyme was subjected to hydroxyapatite chromatography on Ultragel-HA. The column (1.0 x 18 cm) was pre-equilibrated with distilled water and the lipase was applied. Non-binding material was eluted by washing the column with 2 bed volumes of distilled water. Lipase activity was eluted following the application of a linear gradient of distilled water and 0.2 M potassium phosphate buffer pH 6.8. Fractions (4 ml) were collected and assayed for lipase activity as described for the previous step. Active fractions were pooled, dialysed against 10 mM MOPS pH 6.8, containing 1 mM EDTA (1 l) and concentrated by ultrafiltration to a volume of ~5 ml. The purified lipase was stored frozen as beads in liquid nitrogen.

Chromatography on Sephacryl S200. In the case of V205L, the enzyme obtained after the previous step contained a minor impurity as judged by SDS–PAGE. This preparation was subjected to an additional step comprising gel permeation chromatography on Sephacryl S200HR. The concentrated V205L sample was applied to a column of Sephacryl (1.0 x 70 cm) equilibrated in 30 mM MOPS pH 6.8 containing 150 mM KCl and 1 mM EDTA. Fractions (2 mL) were collected and analysed for lipase activity. Active fractions were pooled, dialysed, concentrated and frozen as described in the previous step.

Enzyme assays

Resorufin ester assay. Lipase activity was determined quantitatively using 1,2-O-dilauryl-rac-glycero-3-glutaric acid, resorufin ester as substrate. The assays were performed as suggested by Boehringer, using an absorption coefficient for resorufin of 60.0 l mmol–1 cm–1 at 572 nm. Units were defined as micromoles product formed per min, and specific activity as micromoles product formed per min per mg protein.

Tributyrin/bromocresol purple assay. Column fractions were assayed. A solution containing 5 mM Tris base, 100 mM KCl, 0.5% (w/v) Triton X-100 and 0.04% bromocresol purple was adjusted to pH 7.5 using 1 M NaOH. To 50 ml of this solution was added an equal volume of distilled water followed by 1 ml of tributyrin. Prior to assaying column fractions, the tributyrin/indicator solution was stirred vigorously and an aliquot (150 µl) applied to a well of a 96 well microtitre plate. Subsequently a sample (10–40 µl depending on its activity) of each column fraction was added to a well containing the indicator/tributyrin substrate. Relative amounts of lipolytic activity in samples was judged by whether the indicator in the corresponding well turned yellow from purple and over what time scale this change occurred.

Analytical methods

Determination of protein concentration. The protein content of enzyme samples was determined using the method of Lowry et al. (1951) and employing BSA as a protein standard. Concentrations of BSA were determined by using a value of 6.67 for A279 for a 1% aqueous solution (Janatova et al., 1968Go).

SDS–PAGE. Electrophoresis was carried out using a Pharmacia EPS 600 power supply and Xcell II Mini-Cell apparatus (Novex-Electrophoresis GmbH, Frankfurt-am-Main, Germany). SDS–PAGE was carried out using Novex 10–20% Tricine gels using the manufacturer's recommended conditions. The markers used in determining molecular weights were Novex's Mark 12 wide range standards. Gels were routinely stained using Comassie Brilliant Blue R250.

Western blotting. Western blots were carried out using the Novex blotting module according to the manufacturer's protocol onto PVDF membranes. After transfer the membrane was blocked using 0.5% (v/v) Tween-20 in phosphate buffered saline (PBS) for 1 h at 37 C. The membrane was transferred to a fresh container containing a 1000x dilution of polyclonal mouse anti-RML in 0.05% (v/v) Tween-20 in PBS (PBST) and incubated at 37°C for 1 h and then washed three times in PBST. The membrane was transferred to a fresh container containing a 1000x dilution of alkaline phosphatase labelled rabbit anti-mouse IgG serum (Sigma, UK) in PBST and incubated at 37°C for 1 h before being washed three times in PBST. The blot was developed by the addition of a solution of 0.15 mg/ml 5-bromo-4-chloro-3-indoyl phosphate, 0.30 mg/ml nitro blue tetrazolium, 100 mM Tris–HCl pH 9.5, 5 mM MgCl2 (Sigma, UK). The blot was incubated in this solution at room temperature until blue bands were visible. The development was stopped by rinsing the membrane in distilled water and air drying.

Characterization of recombinant lipases

Enzyme immobilization. The recombinant enzyme purified as described above was `absorbed' to the weak ion-exchange resin (Duolite ES, 568, Rohm & Haas, France), the same support which is used commercially for the immobilization of Rhizomucor miehei lipase (Lipozyme®, Novo-Nordisk A/S, Denmark). For the wild-type, an enzyme solution (2.7 mL, 0.9 mg) in 10 mM MOPS pH 6.8 containing 1 mM EDTA was added to 75 mg of dried Duolite beads and the resulting suspension was shaken at 4°C in an orbital shaken for 4–6 h, or until all the enzyme activity had disappeared from the solution. This, and the increase in lipase activity of the beads, was followed by the resorufin assay. Immobilization of the two mutants was carried our as described above but with 3.6 ml (0.6 mg) of V205l or 4 ml (0.3 mg) of V205I being added to 50 mg of Duolite. The immobilized lipase preparations, and a sample of beads treated without enzyme under the same conditions (control), were dried overnight under vacuum over P2O5.

Preparation of substrates. The substrates were synthesized according to published procedures. The tert-butyloxazolinone 1a was synthesized from tert-leucine via a Schottman-Bauman reaction as outlined in Turner et al. (1995), and iso-propyl oxazolinone 1b was prepared from N-benzoyl-L-valine as described in Di Bello et al. (1971). The NMR spectra of the products obtained were recorded on a Jeol EX270 spectrometer and were in accordance with those reported previously.

Enzymatic reactions. The enzymic ring-opening in the presence of butanol was carried out as described by Turner et al. (1995) with minor modifications. Typically the reaction mixture contained substrates 1a or 1b (18–21 mg), immobilized enzyme (20–24 mg), n-butanol (20 µl) and trace amount of triethylamine in toluene (1 ml). The reactions were carried out in a Luckham R-300 shaker-incubator at 30°C in 2 ml screw capped glass vials at a shaking rate of 150 r.p.m. Samples were withdrawn at regular intervals and, after the immobilized enzyme had settled, an aliquot (50 µl) was removed and diluted ten fold with toluene for GC analysis, which was used to determine the overall rate of conversion and the enantiomeric purity of the substrates and products.

GC analysis. Samples were analysed on a Hewlett–Packard 5890 GC equipped with a flame ionization detector, Chiraldex CB chiral column (25 m longx0.25 µm film thickness x0.32 mm internal diameter; Chrompack, Walton-on-Thames, U.K.) and an autosampler. Aliquots (1 µL) were injected on the column and an isothermal program was used to separate the components. The column temperature was maintained at 160 °C and 140 °C and the run time was 50 and 40 min for the reaction mixtures obtained with 1a and 1b respectively. The injector and detector were maintained at temperatures of 225 °C and 220 °C respectively. Hydrogen was used as carrier gas at a flow rate of 1 ml/min. GC data were analysed using the HPCHEM software (Hewlett–Packard, UK).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
Molecular modelling studies have suggested that the enantioselectivity of RML in the ring opening of oxazolinones 1 (Figure 1Go) may, in part, be due to the difference in steric constraints upon each substrate during binding (Yagnik, 1997Go). The RML complexes with oxazolinones (R)-1a, (S)-1a, (R)-1b and (S)-1b were minimized prior to running molecular dynamics (MD) simulations, in order to optimize the structures and remove any initial bad contacts. Although MD studies in the region of hundreds of ps are fast becoming standard, much information may be gathered from smaller timescales. In this case, the simulation stage of 10 ps was chosen to allow the initial stages of side chain movement and re-orientation of hydrogen bonds to be studied, since most of these are developed within this time period (McCammon and Harvey, 1987). The use of a distance-dependent dielectric was a compromise between full MD studies involving explicit solvent molecules (toluene), which would have greatly increased the calculation times, and employing a constant dieletric of 2.4 representing toluene. Inaccuracies would have resulted from the latter since the active site of RML is at the enzyme-solvent interface. The switching function range was set between 14–15 Å in order to avoid possible discrepancies arising from overlap with the bounday region of harmonic constraints, fixed at 12 Å from the Ser 144 residue. The results of the MD studies showed the rmsd values to be relatively good indicators of the preferred protein-oxazolinone complex conformations (Table IGo).


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Table I. The results from an MD simulation of tetrahedral structures RML-(R)- and RML-(S)-1a, and RML-(R)- and RML-(S)-1b.
 
Visual analysis of the MD time-averaged RML-oxazolinone complexes showed that in the case of RML-(R)-1a the oxazolinone oxygen atom O6 (Figure 1Go) formed a hydrogen bond with the NH group of only one of the oxyanion stabilizing residues, Ser 82 (Figure 2aGo), and another with the OH group of Tyr 28. The hydrogen bonding pattern between the catalytic residues Asp 203 and His 257, as well as oxazolinone oxygen atom O3 (Figure 1Go) and His 257, were as expected for this tetrahedral intermediate complex. In structure RML-(S)-1a, the oxazolinone oxygen atom O6 formed hydrogen bonds with both Ser 82 and Leu 145, but not Tyr 28 (Figure 2bGo). As before, the rest of the hydrogen bonding pattern was as expected. The rmsd for the protein only in these complexes (Table IGo), indicated that the enzyme had to shift more in trying to accommodate the tert-butyl group of RML-(R)-1a into a large hydrophobic pocket, than that of RML-(S)-1a. This resulted in loss of hydrogen bonding between RML-(R)-1a:O6 and the amide group of Leu 145, which was replaced by one from the nearby Tyr 28 hydroxyl group.



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Fig. 2. Hydrogen bonding between O6 of the oxazolinone and nearby enzyme residues in the RML-(R)-1a (a), RML-(S)-1a (b), RML-(R)-1b (c) and RML-(S)-1b (d), all following MD.

 
The carbonyl oxyanion of both iso-propyl-substituted oxazolinones, RML-(R)-1b (Figure 2cGo) and RML-(S)-1b (Figure 2dGo), showed the same hydrogen bonding pattern as RML-(S)-1a (Figure 2bGo). Taking into consideration the small difference in rmsd of the protein following MD, this suggested that both RML-oxazolinone complex conformations were almost equally accessible. The result was therefore in accordance with experimental data on the RML-catalysed biotransformations of 1a and 1b (Winterman, 1996Go).

A plot of the variation in hydrogen bonding distances during the MD simulations allowed a clearer picture of the interactions to emerge. In the case of the tert-butyloxazolinones 1a, the most stable hydrogen bond was between RML-(R)-1a:O6 and Ser 82:NH, followed by Leu 145:NH (Figure 3aGo). The large fluctuation in hydrogen bonding between RML-(R)-1a:O6, Leu 145:NH and Tyr 28:OH (Figure 3aGo) was not seen for RML-(S)-1a (Figure 3bGo). Similar observations on the relative stabilities of these hydrogen bonds and potential role of Tyr 28 in stabilizing the oxyanion intermediate have also been made by Norin and co-workers during a 350 ps MD study on the binding of (R, S)-1-phenyl ethyl hexanoate and (R, S)-2-octyl hexanoate to RML (Norin et al., 1994Go). This demonstrated that a relatively short MD time-scale could nonetheless provide valuable data.



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Fig. 3. Variation in hydrogen bonding distance between protein residues RML-(R)-1a:O6 (a), RML-(S)-1a:O6 (b), RML-(R)-1b:O6 (c) and RML-(S)-1b:O6 (d).

 
Bonding distance variation plots for RML-(R)- and RML-(S)-1b again showed that the hydrogen bond between (R)- and (S)-1b:O6 and Ser 82:NH to be the most stable (Figure 3cGo and Figure 3dGo respectively). The RML-(S)-1b complex showed similar results to that seen for RML-(S)-1a (Figure 3bGo), forming a fairly stable hydrogen bond with Leu 145:NH and intermittently, some favourable interactions with Tyr 28:OH (Figure 3dGo). Intermediate RML-(R)-1b gave comparable results with a similar average bonding distance between (R)-1b:O6 and Tyr 28:OH, though involving higher levels of fluctuation. These graphs were expected to be even more similar due to the observed lack of enantioselectivity in the biotransformation reaction (Winterman, 1996Go). It is possible however, that such differences may have arisen from slight differences in the minimized structures prior to the MD run. Also, the larger fluctuations in RML-(R)-1b could help explain the observed differences in potential energy and rmsd of protein, compared to RML-(S)-1b.

A molecular surface calculation allowed clearer visualization of each RML-oxazolinone complex. In the case of RML-(S)-1a the tert-butyl group fits `snugly' into a large hydrophobic pocket within the active site groove (Figure 4aGo). For the same substituent in the corresponding (R) isomer to lie within this pocket, the protein was forced to adopt a changed conformation, thus making it the less preferred substrate in terms of enantioselectivity. In the case of RML-(R)- and RML-(S)-1b, the iso-propyl group was much less constricted in the hydrophobic pocket and each conformation could be formed with minimal change in the protein conformation (Figure 4bGo). These observations supported the earlier stated hypothesis into the origins of enantioselectivity in this biotransformation.



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Fig. 4. Complementarity between molecular surfaces of enzyme and oxazolinone in RML-(S)-1a (a) and RML-(S)-1b (b) Note that the tert-butyl group in the substrate fits `snugly' into a hydrophobic pocket in active site groove, whilst the iso-propyl group is comparatively less constrained. (c): a Ligplot map showing the major interactions between RML-(R)-1a and the residues within the active site groove and (d) the shift in (R)-1b on mutating Val 205 to Leu, compared to the opposite stereoisomer (S)-1b.

 
It was reasoned that appropriate mutations within one or more of the residues comprising the hydrophobic pocket should affect the biotransformation reaction. 2-D Ligplot (Wallace et al., 1995Go) representations of the inter-residue contacts showed the tert-butyl group of both (R)- and (S)-1a had hydrophobic contacts with Asp 91, Pro 177 and Val 205 (Figure 4cGo). In comparison, the iso-propyl group of (R)-1b interacted with Val 205 and that of (S)-1b formed contacts with Trp 88 (data not shown). Val 205 was therefore chosen for further computational studies, examining the possible effect of mutation to Leu (LeuRML) and Ile (IleRML).

The molecular surface calculations (Figures 4a & bGo) and Ligplot results (Figure 4cGo) had also shown the phenyl ring of substrates 1a and 1b formed hydrophobic contacts with Leu 258 and Gly 266 of RML. This suggested these residues may play a part in directing the stereoselectivity of the enzyme and should perhaps be considered for mutational analysis. However, since the phenyl ring of these oxazolinones was a constant factor in all substrates, it was more likely that the residues interacting with the tert-butyl and iso-propyl substituents were responsible for guiding the observed stereoselectivity. Molecular modelling studies by Scheib et al. (1999) have suggested Leu 258 plays a role in guiding the stereoselectivity of Rhizopus oryzae lipase (ROL) for triracylglycerols (sn-2 substituted triacylglycerols) and mutation of this residue has allowed modification of stereoselectivity (Scheib et al., 1998Go). Subsequent modelling studies have proposed that the stereoselectivities of the Mucorales lipase family (of which RML and ROL are both members) for triradylglycerols is determined by Leu 258 and Gly 266 (Scheib et al., 1999Go). The stereoselectivity of RML for oxazolinones 1a and 1b, since they are radically different from the triradylglycerol structures, would therefore not be expected to be determined by Leu 258 and Gly 266. Nonetheless the observations made by Scheib and co-workers about these residues lent further credence to the results from the current modelling work.

The appropriate tetrahedral complexes for each mutant were built and minimized using standard procedures (see Materials and methods). The V205L mutation, LeuRML-(R)- and LeuRML-(S)-1b, caused a much larger shift in (R)-1b than its opposite stereoisomer (Table IIGo) (Figure 4dGo). In particular, the oxyanion of LeuRML-(R)-1b had moved by approximately 1 Å, resulting in loss of hydrogen bonding with Leu 145:NH and a subsequent gain with Tyr 28:OH. This was similar to the scenario observed during the MD simulation of RML-(R)-1a (Figure 2aGo and Figure 3aGo). In contrast, the V205I mutation resulted in only slight conformational change within IleRML-(R)-1b and none at all in IleRML-(S)-1b (Table IIGo). Although energy comparisons between different chemical species are not normally meaningful, in this case (between LeuRML and IleRML in Table IIGo) they were comparable since Leu and Ile are conformational isomers containing the same number of atoms. Based upon these findings, it seemed possible that the mutation V205L in RML would provide an engineered enzyme which could be used for the stereospecific butanolysis of (±)-1b to yield ester (S)-2b with a measureable enantiomeric excess. In addition, the modelling suggested that using the RML mutant V205I for the same biotransformation may similarly result in an enantiomeric excess of (S)-2b, though perhaps with reduced specificity.


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Table II. Results from a minimization of the mutant LeuRML-(R)- and LeuRML-(S)-1b, and IleRML-(R)- and IleRML-(S)-1b.
 
In order to verify these predictions two recombinant lipases, V205L and V205I, were constructed by `megaprimer' PCR mutagenesis. A degenerate oligonucleotide was used allowing the generation of both mutations simultaneously. Clones were selected and sequenced to confirm the correct sequence was present, before being transformed into Pichia pastoris.

The recombinant enzymes were purified to homogeneity by chromatography as described in Materials and methods (Table IIIGo) and the purity of the enzyme was confirmed by SDS–PAGE and Western blot. The enantioselectivity of the constructs was then compared to that of the wild type enzyme (Figures 5 and 6GoGo). It is evident from the data obtained that as expected the introduction of the bulkier leucine and isoleucine residues led to a significant decrease in the enzyme activity towards 1a (Figure 5Go). The key test however was the rate, and crucially, the enantioselectivity of ring opening of the oxazolinone 1b. In this case there was also a decrease in the specific activity of the lipase but the stereoselectivity appeared to be unaffected by mutagenesis (Figure 6Go).


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Table III. Purification of the wild type and the recombinant lipases.
 


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Fig. 5. Kinetics of the ring opening of the tert-butyl oxazolinone, 1a. Peak areas on the y-axis was obtained from the gas-chromatogram.

 


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Fig. 6. Kinetics of the ring opening of the iso-propyl oxazolinone, 1b. Open and closed symbols refer to the (R)-and (S)-substrates respectively. Peak area on the y-axis was obtained from the gas-chromatogram.

 
It should stressed that this result was not totally unexpected perhaps due to our inability to model such important factors as the effect of solvent (toluene), and nucleophile (butanol), both of which are known to have large effects upon enzyme stereoselectivity and activity (Wescott and Klibanov, 1994; Gubicza and Szakács-Schmidt, 1994Go; Carrea et al., 1995Go). Similarly, the effects of micro-solvent (triethylamine) could not be modelled, though experimental studies have shown it affects the main catalytic process and even the enantiomeric excess ratio (Reslow et al., 1992Go; Triantafyllou et al., 1993Go; Turner et al., 1995Go; Parker et al., 1998Go). Although such shortcomings in modelling are to be expected until these factors can be accounted for, it is also clear that there are limitations to using the simplified, static active site protein models when guiding computer generated re-engineering on such a fine scale, even though the hypotheses into the origins of enyzme enantioselectivity may be correct. It is likely that the mutant enzymes, due to the inherent flexibility and mobility of such systems, were able to accommodate the conformational changes on binding both (R)- and (S)-1b with equal ease, though with decreased activities due to the mutations. This would explain the experimentally observed decrease in activity and lack of enantioselectivity. The results also demonstrated potential limitations that may arise in molecular modelling from using such minimized-only mutant RML-oxazolinone structures. Instead, it is possible that a long MD run may have highlighted any conformational changes in the enzymes.

Importantly, however, the results for the ring opening of 1a showed that the predicted mutations did not eliminate enatioselectivity in the case of the tert-butyl oxazolinone. Since the small size of the iso-propyl substitutent in 1b may not have allowed for the mutations to efficiently favour one enantiomer over the other, it is possible that choosing a larger and more flexible substituent, such a benzyl group, may still show that these mutations have the desired effect depending upon substrate size (Turner et al.,1995Go; Winterman, 1996Go).


    Conclusions
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
The prime objective of this work was to examine the validity of a simple `steric hindrance' model as a basis for stereoselectivity of the lipase-catalysed ring opening of oxazolin-5(4H)-ones. Such models have become increasingly popular in recent years as a means of explaining the enantiomeric preference of a particular enzyme in a set of homologous reactions. Moreover, these models may also offer an immense predictive power to fine tune the selectivity of the enzyme by a relatively straight forward genetic manipulation. As mentioned in the introduction, this approach is especially attractive for large scale industrial applications where the choice of biocatalyst is often limited by the availability of enzyme and its numerous other properties in addition to enantioselectivity. However, although the theories explaining the stereoselectivity of RML in these biotransformations may be valid, this work demonstrates the inherent difficulty in extending such information to re-engineering protein function. It is quite likely that in order to obtain such desired change in stereoselectivity, a combination of mutations and/or varying the reaction conditions may be necessary. Although it is difficult to generalize on the basis of a single example, this feasibility study indicates that either a simple active site box model is not necessarily a good guide for engineering enzymes with the desired enantioselectivity, due to the ability of proteins to structurally `compensate' for the intended conformational effect(s), or other factors highlighted above must be taken into account in the calculations and ultimately in the design. In the authors' view the latter possibility is more likely and the accurate consideration of such components in computational studies represents real challenges for the future.


    Notes
 
3 Present address: IRBM `P. Angeletti', Via Pontina km 30600, 00040 Pomezia (Roma), Italy. Back

4 To whom correspondence should be addressed: Evgeny N. Vulfson; Telephone: +44 1189 357208; Fax: +44 1189 267917;E-mail: jenya.vulfson{at}bbsrc.ac.uk Back


    Acknowledgments
 
The authors are grateful to BBSRC for the financial support of this work and Steve Davies for preparing the oxazolinones for this investigation.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
Andersson,E.M., Larsson,K.M. and Kirk,O. (1998) Biocatal. Biotransform., 16, 181–204.[ISI]

Bernstein,F.C., Koetzle,T.F., Williams,G.J., Meyer,E.E.,Jr., Brice,M.D., Rodgers,J.R., Kennard,O., Shimanouchi,T. and Tasumi,M. (1977) J. Mol. Biol., 112, 535–542.[ISI][Medline]

Bjorkling,F., Godtfredsen,S. E. and Kirk,O. (1991) Trends Biotechnol., 9, 360–363.[ISI]

Brady,L., Brzozowski,A.M., Derewenda, Z.S., Dodson,E., Dodson,G.G., Tolley,S., Turkenburg, J.P., Christiansen,L., Hugejensen,B., Norskov,L., Thim,L. and Menge,U. (1990) Nature, 343, 767–770.[ISI][Medline]

Brzozowski,A.M., Derewenda,U., Derewenda,Z.S., Dodson,G.G., Lawson,D.M., Turkenburg, J.P., Bjorkling,F., Hugejensen,B., Patkar,S.A. and Thim,L. (1991) Nature, 351, 491–494.[ISI][Medline]

Burgess,K. and Jennings,L.D. (1991) J. Am. Chem. Soc., 113, 6129–6139.[ISI]

Carrea,G., Ottolina,G. and Riva,S. (1995) Trends Biotechnol., 13, 63–70.[ISI]

Carriere,F., Withers-Martinez,C., van Tilbeurgh,H., Roussel,A., Cambillau,C. and Verger,R. (1998) Biochim. Biophys. Acta, 1376, 417–432.[ISI][Medline]

Chen,C.-S. and Sih,C.J. (1989) Angew. Chem. Int. Ed. Engl., 28, 695–707.[ISI]

Derewenda,Z.S.,Derewenda,U. and Dodson,G.G. (1992) J. Mol. Biol., 227, 818–839.[ISI][Medline]

Di Bello,C. Felira,F. and D'Angeli,F. (1971) J. Org. Chem., 36, 1818–1820.[ISI]

Faber,K. and Franssen,M. C. R. (1993) Trends Biotechnol., 11, 461–470.[ISI][Medline]

Faber,K., Griengl,H., Hoenig,H., and Zuegg,J. (1994) Biocatalysis, 9, 227–239.[ISI]

Grochulski,P., Bouthillier,F., Kazlauskas,R.J., Serreqi,A.N., Schrag,J.D., Ziomek,E. and Cygler,M. (1994) Biochemistry, 33, 3494–3500.[ISI][Medline]

Gubicza,L. and Szakács-Schmidt,A. (1994) Biocatalysis, 9, 131–143.[ISI]

Haeffner,F., Norin,T. and Hult,K. (1998) Biophys J., 74, 1251–1262.[Abstract/Free Full Text]

Holmquist,M., Haeffner,F., Norin,T. and Hult, K. (1996) Protein Sci., 5,83–88.[Abstract/Free Full Text]

Jaaskelainen,S., Wu,X.Y., Linko,S., Wang,Y., Linko,Y.Y., Teleman,O. and Linko,P. (1996) Ann. N.Y. Acad. Sci., 799, 129–138.[Medline]

Janatova,J., Fuller,J.K. and Hunter,M.J. (1968) J. Biol. Chem., 243, 3612–3622.[Abstract/Free Full Text]

Kazlauskas,R.J. (1994) Trends Biotechnol., 12, 464–472. [published erratum appears in Trends Biotechnol (1995) 13 195].

Kazlauskas, R.J. and Bornscheuer,U.T. (1998). In Kelly, D.R. (ed) Biotechnology vol 8a Biotransformations. 2nd edn. Wiley-VCH, Weinheim, pp. 37–191.

Kazlauskas,R.J., Weissfloch,A.N.E., Rappaport,A.T. and Cuccia,L.A. (1991) J. Org. Chem., 56, 2656–2665.[ISI]

Klibanov,A.M. (1989) Trends Biochem. Sci., 14, 141–144.[ISI][Medline]

Laskowski,R.A., MacArthur,M.W., Moss,D.S. and Thornton,J.M. (1993) J Appl. Crystallogr., 26, 283–291.[ISI]

Lawson,D.M., Brzozowski,A.M., Rety,S., Verma,C. and Dodson, G.G. (1994) Protein Eng., 7, 543–550.[ISI][Medline]

Lemke,K., Lemke,M. and Theil,F. (1997) J. Org. Chem., 62, 6268–6273.[ISI]

Lowry,O.H., Rosebrough,N.J., Farr,A.L. and Randall,R.H. (1951) J. Biol. Chem., 193, 265–275.[Free Full Text]

McCamon,J.A. and Harvey,S.C. (1987) Dynamics of Proteins and Nucleic Acids. Cambridge University Press, Cambridge, UK.

Naemura,K., Fukuda,R., Murata,M., Konishi,M., Hirose,K. and Tobe,Y. (1995) Tetrahedron: Asymmetry, 6, 2385–2394.[ISI]

Naemura,K., Murata,M., Tanaka,R., Yano,M., Hirose,K. and Tobe,Y. (1996) Tetrahedron: Asymmetry, 7, 1581–1584.[ISI]

Norin,M., Haeffner,F., Achour,A., Norin,T., and Hult,T. (1994) Protein Sci., 3, 1493–1503.[Abstract/Free Full Text]

Parker,M.C., Brown,S.A., Robertson,L. and Turner,N.J. (1998) Chem Commun., 20, 2247–2248.

Provencher,L. and Jones,J.B. (1994) J. Org. Chem., 59, 2729–2732.[ISI]

Reslow,M., Adlecreutz,P. and Mattiasson,B. (1992) Biocatalysis, 6, 307–318.

Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: a Laboratory Manual 2nd edition. Cold Spring Harbor Laboratory Press. USA

Santaniello,E., Ferraboschi,P. and Grisenti,P. (1993) Enzyme Microb. Technol., 15, 367–382.[ISI]

Sarkar,G. and Sommer,S.S. (1990) BioTechniques, 8, 404–407.[ISI][Medline]

Scheib,H., Pleiss,J., Stadler,P., Kovac,A., Potthoff,A.P., Haalck,L., Spener,F., Paltauf,F. and Schmid,R.D. (1998) Protein Eng., 11, 675–682.[Abstract]

Scheib,H., Pleiss,J., Kovac,A., Paltauf,F. and Schmid,R.D. (1999) Protein Sci., 8, 215–221.[Abstract]

Tanaka,M., Yoshioka,M. and Sakai,K. (1993) Tetrahedron: Asymmetry, 4, 981–996.[ISI]

Theil, F. (1995) Chem. Rev., 95, 2203–2227.[ISI]

Toone,E.J., Werth,M.J. and Jones,J.B. (1990) J. Am. Chem. Soc., 112, 4946–4952.[ISI]

Triantafyllou,A.O., Adlecreutz,P. and Mattiasson,B. (1993) Biotechnol. Appl. Biochem., 17, 167–179.[ISI][Medline]

Turner,N.J., WintermanJ.R., McCague,R., Parratt,J.S. and Taylor,S.J.C. (1995) Tetrahedron Lett., 36, 1113–1116.[ISI]

Uppenberg,J., Ohrner,N., Norin,M., Hult,K., Kleywegt,G.J., Patkar,S., Waagen,V., Anthonsen,T. and Jones,T.A. (1995) Biochemistry, 34, 16838–16851.[ISI][Medline]

Vulfson,E.N. (1994) In Woolley,P. and Peterson,S.B. (eds.) Industrial Applications of Lipases. Cambridge University Press, Cambridge, UK, pp. 271–288.

Wallace,A.C., Laskowski,R.A. and Thornton,J.M. (1995). Protein Eng., 8, 127–134.[Abstract]

Westcott,C.R. and Klibanov,A.M. (1994) Biochim. Biophys. Acta, 1206, 1–9.[ISI][Medline]

Winterman,J.R. (1996) Chemo-enzymatic methods for the synthesis of optically active amino acids. Ph.D. Thesis, University of Exeter, UK.

Yagnik,A.T. (1997) Molecular modelling applications in rational drug design and the study of enzyme-ligand interactions. Ph.D. Thesis, University of Exeter, UK.

Yagnik,A.T., Littlechild,J.A. and Turner,N.J. (1997) J. Computer-Aided Molecular Design, 11, 256–264.[ISI][Medline]

Received May 26, 2000; revised February 1, 2001; accepted February 15, 2001.





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