1 Research School of Chemistry, Australian National University, GPO Box 414, Canberra, ACT 2601 and 2 CSIRO Entomology,Canberra, ACT 2601, Australia
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Abstract |
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Keywords: Agrobacterium/bioremediation/carboxylated lysine/directed evolution/organophosphates/phosphotriesterase
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Introduction |
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Much work has been done to characterize the structure and function of OPH (Raushel, 2002). It has a structure and active site that are related to other enzymes, yet it acts specifically on phosphotriesters. OPH folds into a TIM barrel, (
ß)8, with a binuclear metal binding site located at the C-terminal end of the barrel. It is one of a superfamily of metal-containing amidohydrolases (Holm and Sander, 1997
). Other family members for which crystal structures are also available include urease (Jabri et al., 1995
), dihydroorotase (Thoden et al., 2001
) and adenosine deaminase (Wilson and Quiocho, 1993
). Like OPH, most of the other enzymes in the superfamily contain two metals in the active site. The activity of OPH in the presence of various metals has been examined with highest activity recorded with Co2+. Structures of OPH have been determined with Zn2+, Cd2+, Mn2+ and Zn2+/Cd2+ in the active site (Benning et al., 2001
) as well as the apoenzyme (Benning et al., 1994
). The enzyme acts on a broad range of substrates. Some substrates are turned over at near diffusion rates whereas others are processed at more modest rates. Structures of OPH have been obtained with the inhibitors diethyl 4-methylbenzylphosphonate, diisopropyl methyl phosphonate and triethyl phosphate (Vanhooke et al., 1996
). These inhibitors are substrate analogues and bind in the active site of the enzyme giving clear indications of how the protein binds substrate. Pockets to accommodate the phosphate substituents have been identified. Raushel and co-workers (Chen-Goodspeed et al., 2001a
) have shown that the stereoselectivity depends on the sizes of three pockets known as the small, large and leaving group subsites. Furthermore, they show that stereoselectivity can be enhanced, relaxed or reversed by simultaneous alterations in the sizes of the three subsites (Chen-Goodspeed et al., 2001b
). The structural and kinetic studies have allowed a plausible enzyme mechanism to be proposed; it involves a single in-line displacement attack (SN2) (Lewis et al., 1988
) with the metal-bridging hydroxide ion acting as the nucleophile (Benning et al., 2000
).
The sequences of OPDA and OPH are similar with 90% identity at the amino acids level (Horne et al., 2002). The most significant difference between the two proteins is at the C-terminus where OPDA has an additional 20 residues. The remaining sequence differences occur throughout the protein with some found in the active site. These sequence differences are thought to be responsible for the variation between the substrate specificities of OPH and OPDA; OPDA exhibits higher kcat values for substrates with shorter side chains and can hydrolyse fenthion and phosmet for which OPH has no activity (Horne et al., 2002
).
Given the potential use of phosphotriesterases for the bioremediation of pesticides and nerve agents such as sarin, soman and VX, much work has been undertaken to understand the nature of the mechanism and substrate-binding determinants. The utility of these enzymes to facilitate bioremediation has been demonstrated by experiments in which OPH has been expressed on the surface of Escherichia coli cells and immobilized on a non-woven, polypropylene fabric for use in detoxifying contaminated wastewaters (Mulchandani et al., 1999). However, further progress in bioremediation may well depend on the identification of new enzymes or in tailoring known enzymes to specific requirements. Thus, understanding how nature modifies enzymes to bring about subtle changes in substrate specificity and catalytic properties is of some interest. Towards this end, we report the structure determination of OPDA and comment on the structural basis for the difference in substrate specificity of OPH and OPDA. In addition, we report the results of directed evolution with OPH. Other workers (Cho et al., 2002
) have also applied directed evolution to OPH; however, the method of selection and the consequent results differ from those described here. In our experiments, OPH mutants were selected on their ability to degrade methyl-parathion, methyl-paraoxon and coumaphos-o-analogue (Figure 1
). The coumaphos-o-analogue contains diethyl substituents wherease the others contain dimethyl substituents. Our selection has produced mutants that show improved catalytic efficiency for substrates that contain either diethyl or dimethyl substituents. To test the ability of the evolved enzymes to degrade compounds other than those used in the selection process, the mutant proteins were tested for their ability to degrade demeton S, a compound that contains diethyl substituents. It should be noted that the substrates used in the evolution experiments were all processed more rapidly by OPDA than OPH. Some of the evolved mutants of OPH had activities that were similar to those of OPDA. We comment on the sequence differences between OPH and OPDA and compare these differences with the sequence changes brought about by directed evolution. In effect, we compare the changes brought about by directed and natural evolution.
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Materials and methods |
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Pesticides were purchased from Chem Service. Molecular biology reagents and enzymes were bought from either Roche or Stratagene. Primers (Table I) were obtained from Geneworks. QIAGEN DNA purification kits were used for all DNA purifications unless stated otherwise.
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The E.coli strain DH5 was used for all aspects of the work described. Cells were grown at 30°C. Cell lines were maintained on LB agar plates supplemented with 100 µg/ml ampicillin. Cultures for production of protein for purification were grown in TB media supplemented with 1 mM CoCl2.6H2O and 100 µg/ml ampicillin and shaken at 200 rpm. Mutant libraries were initially grown on LB agar plates containing 14 µM coumaphos-o-analogue (Harcourt et al., 2002
), referred to as indicator plates. Potential positive mutants were grown in LB medium supplemented with 100 µg/ml ampicillin either in 96-well plates and shaken at 100 rpm or in test-tubes and shaken at 200 rpm.
Construction of pCY76
The plasmid pCY76 (par+, bla+, lacZpo, T710tir+) was constructed to over-express constitutively non-toxic genes in E.coli. The heat-inducible protein over-expression plasmid pND706 (Love et al., 1996
), underwent digestion using SstI to liberate a 130 bp fragment containing the
10 translation initiation region (T7
10tir), ribosome binding site (RBS) and multi-cloning cassette DNA fragment (MCS). The T7
10tirRBSMCS fragment was blunted with Pfu DNA polymerase and subsequently sub-cloned into the Pfu DNA polymerase-blunted BamHI/KpnI sites of pMTL22P (Chamber et al., 1998) to form pCY76.
Expression, purification, crystallization and structure determination of OPDA
Expression. opdA
was obtained using PCR from a 4 kb HindIII fragment in pBluescript KS+ obtained from the Agrobacterium genome (Horne et al., 2002). The primers P3 and P4 were used to isolate opdA. The PCR product was restricted with NdeI and EcoRI, ligated into pCY76, then used to transform competent cells. The opdA sequence from the subsequent clone, pSY1, was confirmed via DNA sequencing. The final opdA used in this study encoded 341 amino acid residues (25365; OPH numbers). The putative signal peptide was deleted (amino acid residues 124) and a new start codon added (amino acid residue 25). In addition, base 1089 (cytosine) was deleted to create a C-terminus homologous to that of OPH; in effect, the amino acid residues 366385 were deleted from wt OPDA.
Purification.
OPDA was purified by a procedure modified from that described by Grimsley et al. (Grimsley et al., 1997). No metals were added to the purification buffers. DH5
pSY1 was grown for 40 h, before cells were harvested and resuspended in 50 mM HEPES buffer, pH 8.0. Cells were disrupted with a French Press. The soluble fraction was loaded on to a DEAE Fractogel column at 1 ml/min. OPDA passed through the column. The differences between OPH and OPDA became apparent after the DEAE Fractogel column; OPDA precipitated during the dialysis against phosphate buffer. Because of this problem, the pH of the flow-through containing OPDA was reduced to 7.0 by dialysis against 50 mM HEPES pH 7.0. OPDA was loaded on to an SP-Sepharose column that had been equilibrated against the dialysis solution. Bound OPDA was eluted with
150 mM NaCl using a linear gradient. SDSPAGE analysis of the eluted OPDA showed >95% purity. OPDA was concentrated via ultrafiltration to 8.3 mg/ml for crystallization. The protein for crystallization was stored in 50 mM HEPES pH 7.0, 150 mM NaCl. Protein concentrations were measured by UV absorption at 280 nm. The extinction coefficient for OPDA was calculated as 29 280 M-1 cm-1. The yield of OPDA was
40 mg/l of culture.
Crystals of OPDA. Crystals were formed using vapour diffusion of hanging drops. Crystals for the in-house dataset were initiated from a mixture of 5 µl of protein solution with 5 µl of reservoir solution that consisted of 20% PEG 4000, 0.1 M sodium citrate and 0.2 M ammonium acetate. Crystals for the synchrotron dataset were initiated from a mixture of 5 µl of protein solution which had been dialysed against a solution containing 1 mM CoCl2.6H2O, 50 mM HEPES and 150 mM NaCl, with 5 µl of a reservoir solution that consisted of 10% PEG 4000 and 0.03 M NaH2PO4.
Data collection and structure determination.
Crystals were transferred into a cryobuffer overnight prior to data collection. The cryobuffer was the same as the crystallization buffer with the PEG 4000 concentration raised to 30%. Data were collected from two crystals which were flash cooled to 173°C in a stream of nitrogen gas. An in-house dataset was collected to a resolution of 2.5 Å and a synchrotron dataset was collected to a resolution of 1.8 Å. Data were processed and scaled using the programs DENZO and SCALEPACK (Otwinowski and Minor, 1997). The merging statistics are given in Table II
. The point group was determined to be P321 with a Vm (Mathews, 1968
) of 2.9 Å3/Da for a single molecule per asymmetric unit. Initial phases were obtained from the in-house dataset by molecular replacement using the program AMoRe (Navaza, 1994
) as implemented in the CCP4 suite of programs (Collaborative Computational Project Number 4, 1994
). The atomic coordinates of a monomer of OPH from PDB entry 1hzy.pdb (Benning et al., 2001
) were used as a search model. The correct solution from a rotation function search using X-ray data terms between 25 and 2.3 Å and a Patterson sphere of 23 Å had a correlation coefficient (cc) of 23.6. The closest incorrect solution had a cc of 10.7. The translation function identified the space group as P3121 and the best solution had a cc and R-factor of 71.5 and 0.373, respectively. The best incorrect solution had a cc and R-factor of 32.9 and 0.555, respectively. Rigid body refinement in AMoRE further improved the cc to 75.7 and the R-factor to 0.373. The model displayed good packing geometry when inspected on a graphics terminal.
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An initial model was used without metals or water molecules in the active site and with an unmodified lysine residue at position 169. Sigmaa (Read, 1986) difference electron density maps (2mFoDFc, mFoDFc) indicated the presence of two metal ions, one CO2 group and two water molecules in the active site. The density immediately adjacent to Lys169 was consistent with covalent modification by carboxylation. This has also been seen in OPH and other members of the amidohydrolase superfamily which contain a binuclear metal centre [urease (Jabri et al., 1985), dihydroorotase (Thoden et al., 2001
)] but not in family members where there is a single metal present such as adenosine deaminase (Wilson et al., 1993). However, it was noted that the temperature factors of the carboxyl moiety refined to significantly higher values than the atoms coordinated to it, when introduced to the model at full occupancy. An occupancy of 0.4 produced B-factors of a similar magnitude to neighbouring atoms (
34 Å2). The identity of the bound metals is not known and possible candidates include the divalent cations Mg2+, Mn2+, Co2+, Ni2+, Zn2+ and Cd2+. Reasonable B-factors and difference maps were obtained with both Mg2+ with occupancies of 1.0 and Co2+ with occupancies of 0.4. Similar values would be obtained for any of the other fourth-row ion species listed above, but Co2+ was used because the protein had been expressed in bacteria that were grown in the presence of 1 mM Co2+. Given that the CO2 group of the carboxylated lysine and the metal ions have been shown to be mutually stabilizing (Shim and Raushel, 2000
), it is tempting to consider the latter more likely. This is further supported by the fact that the key residue Arg254 showed two alternative conformations of its side chain with occupancies of 0.4 and 0.6. A second dataset was collected at the BioCARS beamline, BM14D, at the Advanced Photon Source. The protein had been dialysed against a solution of 1 mM Co2+ prior to crystallization. The model obtained from the in-house data was used as an initial model for refinement using the synchrotron data. The same test reflections were excluded plus a further 5% of data from the higher resolution shells. Again standard CNS protocols were used for the refinement. The resulting model refined well with two Co2+ ions in the active site at full occupancy and only a single conformation of the Arg254 side chain.
Directed evolution of OPH and kinetic analysis of mutant proteins
Error-prone PCR.
The error-prone PCR protocol used was modified from Chen and Arnold (1993). The PCR reaction mixture consisted of 200 µM dNTPs, 5U Taq DNA polymerase, 1 µM primers P1 and P2, 10 ng DNA template (pJK33) (Mulbury and Karns, 1989), 5 mM MgCl2, 0.07 µl ß-mercaptoethanol, 5 µl DMSO, 250 µM MnCl2, 10 µl 10x Taq DNA polymerase buffer and distilled H2O to a final volume of 100 µl. The mutation rate was 12 amino acid changes per gene, determined by sequencing opd from 20 random clones per round of screening. The EcoRI/BamHI-digested PCR product was gel extracted, ligated into pUC19 and then used to transform competent cells. Transformed cells were plated on to indicator plates.
DNA shuffling. opd
mutants were shuffled essentially as described (Stemmer, 1994). The shuffled genes are described in the Results section. Primers P1 and P2 were used. The shuffled genes were cloned into the EcoRI/BamHI sites of pUC19, then used to transform competent cells. Transformed cells were plated on indicator plates.
Site-directed mutagenesis. opd H254R was made using the QuikChange protocol according to the manufacturers directions (Stratagene). The template was pJK33. The primers were P5 and P6. opd H254R from transformants was sequenced to confirm the presence of H254R mutation and no others.
Screening.
Colonies with significant fluorescence were picked with a sterile toothpick and grown in 96-well plate format. The overnight cultures were then assayed for their ability to degrade three pesticides. This was achieved by adding 1030 µl aliquots of each culture to the corresponding wells of three 96-well plates that contained 80 µl of reaction mixture. The reaction mixture consisted of either 0.01 mM coumaphos-o-analogue dissolved in 100 mM TrisHCl (pH 7.0) with 8% methanol, 1.5 mM methyl-paraoxon dissolved in 100 mM sodium phosphate (pH 8.0) with 10% methanol or 0.4 mM methyl-parathion dissolved in 100 mM sodium phosphate (pH 8.0) with 10% methanol. The reaction with the coumaphos-o-analogue was monitored with a POLARstar fluorimeter (Harcourt et al., 2002) and the reaction with methyl-parathion and methyl-paraoxon with a Labsystems Multiskan UV/Vis spectrophotometer at 400 nm. Activities were taken from the slopes of the best-fit lines through the data points. Clones exhibiting the highest activities were retested from single colonies grown in 96-well plate format, then in test-tubes. The activities of these cells were confirmed, as before, and glycerol stocks were prepared.
OPH purification.
The methods used to express and purify OPH and the mutated variants used for kinetic measurements were modified from those described by Grimsley et al.(1997). No metals were added to the purification buffers in either the OPH or OPDA preparations. The soluble cell lysate was passed through a DEAE Fractogel column before OPH was prepared for cation-exchange chromatography as referenced. OPH-containing fractions were pooled, concentrated to
2 mg/ml via ultrafiltration, then stored at 20°C in the presence of 30% glycerol. SDSPAGE analysis of the eluted OPH showed >95% purity. Protein concentrations were measured by UV absorption at 280 nm. The extinction coefficient for OPH and the OPH mutant proteins was calculated as 29 160 M-1 cm-1.
Kinetic assays.
The kinetic constants for the three substrates (coumaphos-o-analogue, methyl-paraoxon and methyl-parathion) were determined by varying the concentrations of the substrate with a constant protein concentration. The protein was diluted with the corresponding assay buffer in the presence of 1 mg/ml BSA, which was used to stabilize the diluted protein. For methyl-parathion, the assay buffer contained 100 mM HEPES pH 8.0, 1 mM CoCl2.6H2O, 1% acetone. For methyl-paraoxon, the assay mixture contained 100 mM HEPES, pH 8.0, 1 mM CoCl2.6H2O, 2% methanol. For coumaphos-o-analogue, the assay mixture contained 100 mM TrisHCl pH 7.0, 1 mM CoCl2.6H2O, 1% methanol. For demeton, the assay mixture contained 100 mM HEPES pH 8.0, 1 mM DTNB [5',5'-dithiobis(2-nitrobenzoic acid)] and 1% methanol. The rate of hydrolysis of methyl-paraoxon and methyl-parathion was measured by monitoring the appearance of p-nitrophenolate at 400 nm (400 = 15 000 M-1 cm-1 at pH 8.0) in a 1 cm cuvette. The rate of hydrolysis of coumaphos-o-analogue was determined by measuring the production of chlorferon at 348 nm (
348 = 9100 M-1 cm-1 at pH 8.0) in a 5 cm cuvette. The rate of hydrolysis demeton was measured by following the appearance of 2-nitro-5-thiobenzoate at 412 nm (
412 = 14 145 M cm-1 at pH 8.0) in a 1 cm cuvette. For methyl-parathion, methyl-paraoxon and demeton, the kinetic constants (Vmax and Km) were obtained by fitting the data to the equation
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Results and discussion |
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The soluble portion of OPDA minus the C-terminal extension was expressed and purified and its structure obtained using the OPH structure as a search model. The numbering scheme used to describe OPDA is that of OPH. The refinement statistics for both models are given in Table II. The stereochemistry was checked by the programs PROCHECK (Laskowski et al., 1993
) and WHATCHECK (Hooft et al., 1996
). The Ramachandran plot showed that all residues were in the most favoured region or additionally allowed regions with the exception of Glu159 and Ser61, which fell in the generously allowed region. Glu159 has been an outlier in the Ramachandran plots of all known phosphotriesterase structures. All stereochemical parameters were inside normal ranges or better than expected in the tests performed by PROCHECK. The overall G-factor was +0.4 for both models.
Overall structure
As can be seen in Figure 2, the overall structure of OPDA is very similar to that of OPH. Both molecules form a dimer with the subunits related by essentially the same crystallographic twofold axis. Overlaying all main chain atoms for residues 35361 results in an r.m.s. displacement of 0.38 Å. The TIM barrel region overlays exceedingly well (r.m.s. displacement 0.27 Å). Some small rigid body movements of secondary structural elements are seen away from the barrel but these are still only of the order of 1 Å. The secondary structure is essentially the same as that of OPH and consists of 10 ß-strands, 14
-helices and four 310 helical turns. The active site sits at the C-terminal end of the barrel and comprises residues from secondary structural elements disparate in sequence space. There are a number of helices on the bottom of the barrel. In OPH, one of these helices forms one side of a binding pocket for phenylethanol. The structures of the two opda models were overlayed. The structures showed excellent agreement; r.m.s. displacement over all protein atoms was 0.2 Å. However, there were differences in the conformation of the side chains in certain key active site residues that occurred with the change in metal ion occupancies and the associated conformational change of Arg254. Significant shifts (
1 Å) occurred for the residues Tyr257, Phe272, Leu271, Leu303 and Ser203. The first three of these are located in the large binding pocket sub-site and Leu303 is located in the small binding pocket sub-site. The synchrotron dataset also showed additional +fofc density in the active site. The density was consistent with a HEPES molecule binding in multiple conformations. The sulfonate moiety appears well ordered at full occupancy but the rest of the density is broken and consonant with multiple conformers. A single sulfite ion was located from the HIC-UP database of hetero-compounds and included in the refined model.
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The model derived from the in-house data shows that the two Co2+ ions, and ß, are coordinated in a similar manner to the Zn2+ ions in the OPH active site. The
metal forms bonds with His55, His57 and Asp301 and is more buried than the ß that forms coordinate bonds to His201 and His230. In addition, the two metals are linked by a bridging hydroxide ion and side-chain oxygens of the carboxylated Lys169. The
metal has trigonal bipyramidal coordination whereas ß exhibits distorted trigonal bipyramidal coordination (Figure 3
). In the model derived from the synchrotron data the sulfite ion binds with one of its oxygen atoms replacing the bridging hydroxide ion seen in the other model. Figure 3
also shows the position of both conformers of the neighbouring residues which change between the two datasets. A superposition of the substrate analogue trimethyl phosphate taken from 1eyw.pdb is also shown.
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Structural consequences of sequence differences between OPH and OPDA.
There are about 30 sequence differences between OPH and OPDA (Figure 4). The largest group consists of 19 residues that are spread over the solvent-exposed surface of the molecule. In addition, two regions of the molecule with a significant number of sequence differences were identified. One has already been mentioned; the large pocket of the active site. The other is the region of the protein that is responsible for binding phenylethanol in OPH. This compound resembles one of the reaction products of OPH and might be expected to bind in the active site and function as an inhibitor. Contrary to this expectation, phenylethanol binds to OPH on the exterior of the molecule on the opposite face and removed from the active site cavity. In OPH this site is formed by residues Met293, Lys294, Glu295 and Thr352 that become Lys293, Asp294, Arg295 and Ala352 in OPDA. Apart from the sequence changes, the water structure in this region differs in the two proteins. Given these significant differences, it is unlikely that phenylethanol would bind to OPDA at the same location.
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The object of the directed evolution experiments was to find a series of mutations in OPH that would give it similar activity to OPDA. The starting point of the evolution was a form of the OPH protein that had five additional residues from ß-galactosidase fused to the N-terminus. The gene for OPH was subjected to one cycle of random mutagenesis followed by two cycles of gene shuffling. As part of each cycle, mutants were selected in two stages. In the first stage, the activity of mutants towards coumaphos-o-analogue was monitored on agar plates, whereas in the second stage the activities of mutants towards three substrates, coumaphos-o-analogue, methyl-paraoxon and methyl-parathion, were measured more carefully with a 96-well plate reader.
Error-prone PCR was used to create a library that was initially screened on plates containing 4 µM of the coumaphos-o-analogue. The library gave rise to about 20 000 colonies of which about 80% showed little activity. About 1600 colonies from each library displayed fluorescence that was equal to or better than that of the wild-type enzyme. A 96-well plate reader was then used to measure the ability of these clones to degrade the three substrates. A total of 48 potential positive clones were then used in the first round of DNA shuffling. About 10 000 clones were screened on the LB medium plates containing 2 µM coumaphos-o-analogue. On the basis of their fluorescence signal, the activities of 800 clones towards three substrates were measured with a plate reader. Of these, 62 clones were re-screened as potential positive variants and, of these, 30 clones with higher activity than wild-type were selected for another round of shuffling. The sequences of the genes of three proteins with enhanced activity were determined. All three contained the change of H254R along with other changes. A further round of DNA shuffling was carried out with the best 30 clones from the first cycle of shuffling. In this cycle of shuffling, 10 000 clones screened on the LB medium plate containing 2 µM coumaphos-o-analogue. The activities of 800 clones towards the three substrates were measured. Of these, 51 clones were re-screened as potential positive variants and, from these, 20 clones were used to prepare protein for kinetic characterization.
Kinetic characterization of evolved OPH proteins
The evolved variants of OPH were purified and their kinetic constants measured with three substrates (Table III). One mutant was selected from the first generation (1G) and 11 from the third generation (3G). The sequence changes in all these mutants along with the relevant kinetic data for OPH and OPDA are also given in Table III
. All the proteins displayed substrate inhibition with coumaphos-o-analogue and Ksi was also measured. The kinetic constants previously reported for OPH vary considerably owing to the method of protein purification and the assay conditions. The values for kcat/Km given in the present work are lower than the highest reported, probably owing to the differences in assay conditions. However, the same assay conditions were used throughout this work and give a good indicator of the relative activities of OPH and OPDA.
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All of the mutants from the third generation contain the H254R mutation. However, this mutation only appears to have a significant effect on the kinetic parameters obtained with the coumaphos-o-analogue. The mutation would tend to be retained because the coumaphos-o-analogue is the only substrate used in the first stage of screening. To survive the screening process all mutations must have enhanced activity towards the coumaphos-o-analogue. With this substrate, the H254R mutation causes a substantial drop in Km along with a drop in kcat. The drop in Km is desirable and appears to have been retained by the third generation mutants, while the drop in kcat is undesirable and appears to be compensated for by additional mutations.
Apart from the substrates used for selection, the mutant proteins were tested for their ability to degrade demeton S. As is evident in Table III, this compound is degraded more rapidly by OPDA than OPH, but neither enzyme degrades it with great efficiency. The catalytic properties of the mutant enzymes show a similar trend to those obtained with the other substrates. The Km values for all the enzymes are high and have not been improved by the directed evolution and enhanced activity has been gained by an increase in kcat. The best enzyme (3G32) has a kcat/Km that is similar to that of OPDA. The fact that demeton was not used in the selection process but was degraded with improved efficiency by the mutant enzymes suggests that the selection process maintained the broad specificity of the OPH.
Sequence changes in terms of structure; natural and directed evolution
Directed evolution has been used to evolve OPH into a series of mutant enzymes that have catalytic properties that are similar to those of OPDA. The residues that are altered in the directed evolution experiments are given in Table IV, as are the corresponding residues in OPDA. Three of the mutations, including the most common (H254R), produce the amino acid that is found in the OPDA sequence while one mutation gives rise to an amino acid not found in either OPH or OPDA. The mutations resulting from directed evolution appear to be clustered at specific sites in the protein, as listed in Table IV
and are best discussed in terms of their location in the protein.
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The second set of mutations, I274T and N265D (Figure 5), are found in an exposed loop that follows strand 7 that supports the large sub-site residues His254 and His257. These mutations give a more hydrophilic sequence that is more appropriate for an exposed peptide. One of the mutations, I274T, is found in the OPDA sequence, yet the structure of the peptide in the vicinity of residue 274 is very similar in OPH and OPDA. On the basis of structural evidence alone, neither mutation would be considered as a good candidate to change the activity of the protein. However, as shown in Table III
, there is experimental evidence that one of these mutations, I274T (clone 3G3), does have an effect on activity in the absence of other changes. The loop on which it is situated has the highest B-factors of any peptide in both OPH and OPDA, suggesting that it may be mobile. In OPH, the structure of this loop changes dramatically and becomes partially disordered when metals are removed (Benning et al., 1995
), again supporting the idea that the loop is mobile. These mutations are likely to affect the dynamics and stability of the protein and may not alter the average structure of the protein as determined by crystallography. These mutations may exert an effect on enzymatic activity of the protein through the dynamics of a surface loop. This effect may involve Leu271 or Phe272, whose side chains protrude into the large pocket of the active site. The conformation of these side chains showed movement when compared between the in-house and synchrotron data models. We have already noted that site-specific changes made at position 272 change the substrate specificity of OPDA.
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The fourth set of mutations consist of Q211L, D208G and K185R (Figure 5). These cluster at the external surface between two outer helices which, like the phenylethanol binding site, is somewhat remote from the active site. Two of these mutations, K185R and Q211L, were first detected during the initial random mutagenesis and gave rise to discernible improvements in activity. The sequence and structure of OPH and OPDA are very similar in the region around the mutations. Gln211 forms a hydrogen bond with the backbone of Ala176. This interaction would be lost in the Q211L conversion. Asp208 forms hydrogen bonds with the backbone of Gly174 and, again, this interaction would be lost in the D208G conversion. The conversion of K185R allows the formation of hydrogen bonds to Glu219 or Glu181. The first two mutations cause a loss in the interactions that stabilize the structure while the third potentially adds a stabilizing interaction.
Conclusion
Given the structures and sequences of OPH and OPDA, one would predict that the most direct way of evolving OPH into OPDA would be to alter the residues in the large pocket of the active site. Of the numerous differences between OPH and OPDA in this pocket, only H254R is observed among the directed mutants. This mutant is found in all the most active mutants that were produced after two rounds of shuffling. In OPDA, Arg254 is well placed to interact with the substrate and may have a significant effect on the catalytic properties of the enzyme. This residue is shown to be capable of adopting more than one conformation and the conformational change is associated with movements of the side chains of several residues which form the large sub-site of the substrate binding pocket. Other mutations in the large pocket probably do not occur because the large pocket in OPH is better suited than OPDA to bind the diethyl substituents of the coumaphos-o-analogue. In other words, the selection process has identified the one mutation in the large pocket that improves the catalytic properties of OPH while maintaining its capacity to degrade efficiently substrates with larger alkyl substituents. Despite its prevalence among the directed mutants, the H254R mutation was not found as a single mutation in any clone with high activity. It was noted in the section dealing with directed evolution that the H254R change when combined with other mutations is capable of producing discernible effects on activity, particularly with the coumaphos-o-analogue.
We noted in the previous section that mutations are found in several locations that are remote from the active site. It is difficult to rationalize these changes mechanistically apart from concluding that they synergistically create small structural changes, which affect the dynamics and/or stability of the protein in a way that enhances substrate binding or subsequent catalytic turnover.
One of the aims of this study was to compare the effects of natural and directed evolution on the evolution of OPH into OPDA. In terms of amino acid sequence changes, there were some similarity in the two processes. In both cases, the H254R change was observed. While this was not unexpected, the I274T and the T352A changes were not anticipated. In terms of structure, the locations of mutations are very similar for both natural and directed evolution. In both natural and directed evolution, mutations appeared to be found in three sites. Whereas the changes to the active site can be rationalized, the effects of mutations on a surface loop and the phenylethanol binding site are more difficult to understand. The fact that mutations in these regions were found in both natural and directed evolution argues that these regions have a physical significance that warrants further investigation. While the similarity between natural and directed evolution was surprising, it should be noted that directed evolution did find ways of enhancing activity that did not occur in natural evolution. These mutations, such as D208G, are again remote from the active site. The observation that mutations outside the active site appear to enhance enzyme activity underscores the fact that enzyme activity depends on many factors and that evolutionary objectives can be achieved in many ways.
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Notes |
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Acknowledgments |
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References |
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Received August 9, 2002; revised November 26, 2002; accepted December 3, 2002.