Department of Biotechnology and Molecular Sciences, University of Insubria, via J.H. Dunant 3, 21100 Varese, Italy
1 To whom correspondence should be addressed. E-mail: loredano.pollegioni{at}uninsubria.it
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Abstract |
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Keywords: directed evolution/flavoprotein/functionstructure relationships/substrate recognition
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Introduction |
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In recent years, it has become an important research goal in various fields to be able simply and accurately to determine and quantify D-AA in biological samples owing to the increasing interest in the significance of the presence of these D-isomers when present in various eukaryotic tissues or fluids, and also in food. In the latter case, D-AA (especially D-Ala, D-Asp and D-Glu) are formed during food processing and also originate from microbial sources, including water, soil and other environments, and may become part of the human diet. Both the D-AA content and the D/(D + L) ratio have been proposed as reliable molecular markers of ripening and as indexes for the assessment of food product quality (Gandolfi et al., 1990; Marchelli et al., 1997
). D-AA have been found in milk and cheeses, fruits and vegetables, processed foods, eggs, honey, infants' formula, fish meal, wine, beer, etc. (Friedman, 1999
). The occurrence of D-AA in foods is normally associated with a decrease in protein digestibility, thus affecting the bioavailability of essential amino acids, facilitating the formation of nutritionally antagonistic and toxic products and ultimately impairing the nutritional quality of food. Traditional analytical methods (such as gas chromatography, capillary electrophoresis and high-performance liquid chromatography) are not suitable for the simple and accurate determination and quantification of D-AA in foods and for on-line application (Brückner et al., 1995
; Brückner and Westhauser, 2003
). Many research groups have proposed enzyme sensors for the specific detection of D-isomers of amino acids based on the detection of hydrogen peroxide or ammonia produced by the DAAO reaction (even coupled to horseradish peroxidase or fungal peroxidase) (Kacaniklic et al., 1994
; Riklin et al., 1995
; Sarkar et al., 1999
). The activity of these sensors is based on DAAO from pig kidney and they are essentially active on neutral D-AA. However, their application was initially limited by the low turnover of the mammalian enzyme and the inactivity on acidic D-AA. The first problem was overcome by using a sensitive biosensor produced by adsorbing RgDAAO on a graphite electrode (Sacchi et al., 1998
); the second was recently solved by producing the M213R RgDAAO mutant active on D-Asp (Sacchi et al., 2002
). This mutant enzyme was produced by combining the structural and functional information available on RgDAAO, given by its crystal structure (Umhau et al., 2000
; Pollegioni et al., 2002
) and mutagenesis studies (Harris et al., 1999
; Molla et al., 2000
; Boselli et al., 2002
) and on D-aspartate oxidase (the flavoenzyme that selectively catalyzes the oxidation of acidic D-AA) with the simulated annealing docking of D-Asp at the active site of both enzymes and investigation on substrate recognition (Sacchi et al., 2002
). An
100-fold increase in the catalytic efficiency on D-Asp (expressed as the Vmax/Km ratio) was observed for the M213R single-point mutant, in which a correct orientation of D-Asp in the active site is achieved. In contrast, the catalytic efficiency on neutral D-AA suffered a dramatic decrease (
17-fold) and the DAAO mutant was practically inactive on basic D-AA (Sacchi et al., 2002
). Even so, and because of their substrate specificity, neither wild-type nor M213R RgDAAO are useful for determining the total concentration of D-AA in food samples or for distinguishing between different D-AA.
Directed evolution is a powerful tool for reshaping the functional and structural features of proteins, including their solubility, enzymatic proficiency and specificity. In the past few years there have been reports of the successful alteration of the substrate specificity of several enzymes (Arnold et al., 2001; Zhao et al., 2002
). Here, we report on the application of directed evolution in achieving DAAO variants with different substrate specificities. The ultimate goal is the generation of a family of DAAO proteins for the development of a pilot biosensor (to detect and quantify the total amount of D-AA, and also to determine the amount of neutral and acidic D-AA) that can rapidly determine the amount of D-AA in foods. In addition to obtaining enzyme variants increasingly more suitable for biotechnological applications, the combination of results from structural studies (Umhau et al., 2000
; Pollegioni et al., 2002
), rational design (Sacchi et al., 2002
) and directed evolution experiments should give us insight into the modulation of substrate specificity in this class of (flavo)enzymes and can be used to explore the limits of protein function.
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Materials and methods |
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Restriction enzymes were purchased from New England Biolabs, Taq DNA polymerase from Promega Life Sciences and AmpliTaq DNA polymerase from Applied Biosystem. T4 DNA ligase was obtained from Promega Life Sciences and from Roche. Horseradish peroxidase was purchased from Roche and D-AA, ligands and all other compounds from Sigma. Kinetic experiments were performed in 100 mM sodium pyrophosphate pH 8.5; ligand binding experiments were carried out in 50 mM HEPES pH 7.5, 10% glycerol, 5 mM 2-mercaptoethanol and 0.3 mM EDTA.
PCR-based random mutagenesis
For the first round of error-prone PCR, the pT7-DAAO plasmid (Molla et al., 1998) was used as template and the whole sequence encoding DAAO was chosen as the target of mutagenesis. Two oligonucleotides, 5'-TATAGGGAGACCACAACGGTTTCC-3' and 5'-CTCATGTTTGACAGCTTATCATCGATAAG-3', were used as 5' and 3' primers. The amplified fragments contained NdeI and HindIII restriction sites so that the PCR products could be ligated with the pT7 vector that was digested with the same enzymes (see below). The first round of error-prone PCR was performed using 20 ng of template DNA in 100 µl of reaction mixture under three different sets of conditions: (1) 10 mM TrisHCl pH 8.3, 50 mM KCl, 2 mM MgCl2, 0.25 mM MnCl2, 20 µM each dNTPs, 0.1 µM each of the 5' and 3' primers and 2.5 U AmpliTaq; (2) 10 mM TrisHCl pH 9.0, 50 mM KCl, 0.1% Triton X-100, 10 mM 2-mercaptoethanol, 10% DMSO, 1.5 mM MgCl2, 50 µM each dNTPs, 0.5 µM each of the 5' and 3' primers and 2.5 U Taq DNA polymerase; (3) 10 mM TrisHCl pH 9.0, 50 mM KCl, 0.1% Triton X-100, 7.5 mM MgCl2, 200 µM each dNTPs, 0.1 µM each of the 5' and 3' primers and 2.5 U Taq DNA polymerase. In all cases, PCR amplification was carried out at 94°C for 40 s, 60°C for 30 s and 72°C for 90 s, 40 cycles in total. The second round of error-prone PCR was performed starting from clone 1-7 (Q144R mutant) resulting from the first round of mutagenesis (see below). The cDNA coding for Q144R DAAO mutant was thus sub-cloned into the pT7-HisTag vector (Fantinato et al., 2001
) and used as template for amplification with the oligonucleotides 5'-GGTGCCTCACTGATTAAGCATTGGTA-3' and 5'-CAGCTGCCGATTCGAACTTCTCGATT-3' as 5' and 3' primers. The amplified fragments contained NotI and XbaI restriction sites at which the PCR products could be ligated with the pT7-HisTag vector digested with the same enzymes. The PCR was performed using 20 ng of template DNA in 25 ml of reaction mixture under two different sets of conditions: (4) as under the first set of conditions above, but using 0.2 mM dGTP and dATP, 1 mM dCTP and dTTP and 0.4 µM each of the primers (25 cycles); and (5) as under the third set of conditions (40 cycles). In all cases, the size and yield of the amplified DNA fragments was determined by gel electrophoresis.
Construction of a random mutants library
Enzymatic DNA modifications were carried out according to the manufacturer's instructions and following standard protocols as described by Sambrook et al. (1989). The PCR-generated DAAO fragments were purified and digested with NdeI and HindIII (first round of mutagenesis) and NotI and XbaI (second round of mutagenesis) restriction enzymes. Digested fragments were separated by gel electrophoresis, purified and ligated to a NdeI/HindIII-digested pT7 vector (first round of mutagenesis) (Molla et al., 1998
) and to a NotI/XbaI-digested pT7-HisTag vector (second round of mutagenesis) (Fantinato et al., 2001
). The ligation products were used to transform JM109 and DH5
E.coli cells, respectively, according to a modification of original procedure by Inoue et al. (1990)
. All colonies on the transformation plates were then collected for DNA extraction; 20 ng of the extracted DNA were used to transform competent BL21(DE3)pLysS E.coli expression cells. Single colonies from LB plates containing 100 µg/ml ampicillin and 34 µg/ml chloramphenicol were used to inoculate fresh LB medium and then incubated overnight at 37°C for the screening procedure and to determine the DNA sequence.
Screening for enhanced DAAO substrate specificity
A saturated E.coli culture (300 µl) was induced by adding 1 mM IPTG and then incubating it at 30°C for 2 h; 50 µl of the induced cells were then transferred to three wells (one for each substrate tested). Cells lysis was performed by adding 150 µl of lysis buffer (50 mM sodium pyrophosphate buffer pH 8.5, 1 mM EDTA, 100 mM NaCl, 40 µg/ml lysozyme and 1 µg/ml DNase I) and incubating the plates at 37°C for 30 min. Oxidase activity was assayed on the crude extracts (200 µl) by adding 100 µl of 90 mM substrate (D-Ala, D-Asp or D-Arg), 0.3 mg/ml o-dianisidine and 1 U horseradish peroxidase in 100 mM sodium pyrophosphate buffer pH 8.5. The product of this enzymatic reaction has a strong extinction coefficient at 440 nm. The time course of the absorbance change was followed at room temperature by using a microtiter plate reader (Biotrak II, Amersham Biosciences) and comparing it with that of cells expressing wild-type RgDAAO (for the first round of mutagenesis) and wild-type and Q144R HisDAAOs (for the second round of mutagenesis) as control. Each clone was evaluated in independent trials and the average was calculated; the mutants that outperformed the control were selected and used for further analysis.
Expression and purification of DAAO mutants
The cDNA of DAAO variants selected from the first round of mutagenesis was excited by EcoRI digestion from the plasmid DNA of selected mutants and subcloned into the pT7-HisTag vector that contained an additional sequence, thus yielding a protein with an N-terminal six-histidine tag (Fantinato et al., 2001) and transferred to BL21(DE3)pLysS E.coli cells. The pT7-HisDAAO variants selected from the second round of mutagenesis and screening procedure were instead directly transferred to the E.coli expression strain. The cells were grown overnight at 37°C in LB medium containing 100 µg/ml ampicillin and 34 µg/ml chloramphenicol and induced at different OD600 values (0.8 or 2.5) by adding 0.61 mM IPTG. After the induction, the cells were cultivated at different temperatures (25 or 30°C) for 3 h and then collected by centrifugation. The crude extract was prepared and the DAAO mutants were subsequently purified as previously reported for wild-type His-DAAO (Fantinato et al., 2001
). The expression of the mutants was also investigated by means of western blot analysis, using an immunostaining procedure (Molla et al., 1998
). The molecular mass of the random mutants was determined by gel permeation chromatography on a Superdex 200 column (Amersham Pharmacia Biotech) using 50 mM sodium pyrophosphate pH 8.5, 5% glycerol, 250 mM NaCl as elution buffer.
Activity assay and spectral experiments
DAAO activity was assayed with an oxygen electrode at pH 8.5 and 25°C with 28 mM D-Ala as substrate and at air oxygen saturation ([O2] = 0.253 mM) (Molla et al., 1998). One DAAO unit is defined as the amount of enzyme that converts 1 µmol of D-Ala per minute at 25°C. Substrate specificity was investigated by means of the same polarographic assay, employing different concentrations of various D-AA as substrate. The previously developed amperometric biosensor (Sacchi et al., 1998
) works on a kinetic assay basis, i.e. the current response is directly dependent on the hydrogen peroxide production by the enzymatic oxidation of D-AA. Thus, and in order to identify the DAAO mutants most suitable to quantify the entire content of D-AA, we measured v0, the initial rate of oxygen consumption (that for wild-type DAAO depends strongly on the composition of the assay mixture) (Pollegioni et al., 1992
), using D-AA solutions (10 mM final concentration) containing different proportions of D-Ala (010 mM), D-Glu (08 mM), D-Lys (08 mM), D-Gln (03.3 mM) and D-Met (02 mM). The protein concentration of purified random mutants was determined using the extinction coefficient of wild-type DAAO at 455 nm (12 600 M1 cm1) (Molla et al., 1998
).
The dissociation constant for ligands was measured spectrophotometrically by adding small volumes of concentrated stock solutions to a sample containing 900 µl of 10 µM enzyme at 15°C (Harris et al., 1999
). The change in absorbance on adding ligand was plotted as a function of ligand concentration, after correcting for any volume change. Circular dichroism (CD) spectra were recorded on a Jasco J-810 spectropolarimeter.
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Results |
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The amount of amplification product recovered by mutagenic PCR was usually lower than that obtained under optimal conditions. In particular, in the presence of 0.25 mM MnCl2 (condition 1), a yield of 25% in comparison with native conditions was obtained, whereas under conditions 2 and 3 a higher figure (
50%) was reached. Interestingly, in the second round of mutagenesis using the pT7-HisDAAO-Q144R as template and error-prone PCR conditions 4 and 5, a higher amplification yield was obtained (
80% compared with native conditions). For each set of conditions tested, a library of
104 clones was generated and up to 1200 independent clones obtained for each PCR condition were screened for DAAO activity on different substrates (D-Ala, D-Asp and D-Arg, at a final concentration of 30 mM). In order to estimate roughly the frequency of mutations, three independent clones for each condition were sequenced: frequencies of mutation of 0.77, 0.37, 0.17, 0.15 and 0.25% were determined for conditions 15, respectively. If the mutation frequency is too high, most of the enzymes generated will be inactive; if it is too low, the wild-type background will be too high and diversity too low. The observed mutational frequencies resulted in 27 base pair mutations per mutant in the first round and 15 base pair mutations per mutant in the second round of error-prone PCR. Transition and transversion occurred with equal frequency for all conditions tested during the first round of mutagenesis, whereas the strongest bias towards transition (e.g., AG substitutions) was observed for conditions used in the second round of mutagenesis (Table I). Levels of 91, 27 and 17% of inactive mutants resulted from the first round of mutagenesis under PCR conditions 1, 2 and 3, respectively, whereas the second round of mutagenesis under PCR conditions 4 and 5 gave rise to 63 and 77% of inactive mutants, respectively.
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Directed enzyme evolution requires the rapid and sensitive selection of the properties of interest. The colorimetric assay we used (based on the coupling of the H2O2 produced by DAAO with the reaction of horseradish peroxidase and o-dianisidine) is adequate for determining the alteration of the substrate specificity of RgDAAO. Among the DAAO mutants generated during the first round of mutagenesis under PCR condition 1, a significantly higher activity on D-Ala with respect to the wild-type enzyme was detected in the crude lysate of an E.coli culture expressing mutant clone 1-7 (Figure 1A). Furthermore, three mutants (clones 2-12, 2-41 and 2-381) derived under the second set of PCR conditions and one mutant derived under the third set of conditions (clone 3-382) showing an altered substrate specificity were isolated. As shown in Figure 1B and with respect to the wild-type DAAO, mutants 1-7 and 2-41 retained similar activity on D-Ala but different activity on D-Arg, whereas mutants 2-12 and 2-381 showed lower activity on all substrates tested. In contrast, the activity of the latter mutants on D-Asp and D-Arg is higher than the value determined on D-Ala (compare Figure 1B and C).
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Expression and purification of DAAO random mutants
Selected DAAO variants identified in the first round of mutagenesis were expressed and purified as reported by Fantinato et al. (2001). However, whereas 6 and 3.6 mg of purified protein/liter fermentation broth were obtained for D242V/Q253R/D304V (clone 2-41) and Q144R (clone 1-7) mutant, respectively, a 24-fold lower yield was obtained for the L118H (clone 3-382) mutant (0.25 mg of purified DAAO mutant/liter fermentation broth) and no enzyme was purified following HiTrap chelating chromatography from the crude extracts of D40G/C234R (clone 2-12) and D40G/L135H/Q339L (clone2-381) mutants. Western blot analysis indicates that the low expression yield of these latter mutants is probably due to the formation of inclusion bodies: 8090% of the expressed mutant DAAO protein was in the insoluble fraction after cell lysis and no DAAO proteolytic products were detected in the crude extracts.
A significant increase in the expression yield of the L118H DAAO mutant (clone 3-382) was obtained when the cells were grown overnight at 37°C, induced at OD600 = 2.5 by adding 1 mM IPTG and then cultivated for up to 3 h at 25°C. As a result, 3.6 mg of purified enzyme per liter of fermentation broth were produced.
An increase in enzyme expression for clone 2-381 was obtained by growing the cells at 15°C for up to 48 h. A significantly higher amount of immunoreactively expressed protein was detected in the crude extract by means of western blot analysis; however, in the meantime multiple bands at lower molecular mass appeared, suggesting a susceptibility of the expressed mutant to proteolysis (data not shown). The change in the expression conditions did not improve the enzyme recovery for D40G/C234R and D40G/L135H/Q339L DAAO variants (clone 2-12 and 2-381, respectively), hence further characterization was not feasible.
Concerning the DAAO mutants identified in the second round of mutagenesis, the T60A/K152E/Q144R mutant (clone 4-903) was efficiently expressed under the conditions reported above for wild-type (Fantinato et al., 2001): a figure of 5.5 mg of pure DAAO/liter of fermentation broth was obtained. On the other hand and although different expression conditions were analyzed, the Q142R/G199D/Y223C/H329R mutant (clone 5-249) was not expressed as a soluble and active enzyme. Significant expression of this latter mutant DAAO was only obtained when the His-tag sequence was absent and the protein expressed as detailed by Molla et al. (1998)
(up to 2.1 mg of pure DAAO/liter of fermentation broth). This result suggests a specific interference of the His-tag in the folding process of this mutant DAAO.
Like the wild-type RgDAAO (Molla et al., 1998; Fantinato et al., 2001
), all purified DAAO mutants were >95% homogeneous (as indicated by the E280/E455 ratio of
8.3 for the absorbance spectrum and SDSPAGE analysis), were dimeric holoenzymes (according to gel permeation chromatography using 1 mg/ml protein samples) and were stable for several months when stored at 20°C. Except for mutant 5-249 (which shows a more pronounced ellipticity of the
220 nm peak), far- and near-UV CD spectra of all purified mutants are superimposable.
Kinetic and binding properties of evolved DAAO enzymes
To investigate the substrate specificity of the mutants generated by error-prone PCR and identified by the screening procedure, we determined the activity of wild-type and DAAO variants on different amino acids, measuring the oxygen consumption with a Clark-type electrode at pH 8.5 and 25°C (Molla et al., 1998). The apparent kinetic parameters Vmax and Km on different D-AA measured at a fixed (21%) oxygen concentration are summarized in Table II: D-Ala was chosen as the reference substrate and D-Asp and D-Arg as models of an acidic and a basic D-AA, respectively.
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In order to identify the DAAO variants more appropriate to detect the entire content of D-AA, we determined v0, the initial velocity of the enzyme reaction (expressed as µmol of oxygen consumption/mg protein) on D-AA mixtures (10 mM final concentration) containing different ratios of D-Ala, D-Glu, D-Lys, D-Gln and D-Met, since this parameter is highly dependent on the mixture composition for wild-type DAAO (see Materials and methods). When compared with the value measured on 10 mM D-Ala, the mutant Q114R/G199D/Y223C/H329R showed significantly lower v0 values with all the D-AA mixtures containing a D-Ala concentration <5 mM, while the M213R DAAO showed appreciably lower activity on all mixtures not containing D-Glu. Among the DAAO variants available, the L118H and T60A/Q144R/K152E mutants exhibited the lowest variability of response as function of D-AA composition: a mean value of v0 corresponding to 87 ± 19 and 76 ± 15% of the value determined on 10 mM D-Ala was determined, respectively. These values compare with 63 ± 15, 69 ± 14, 45 ± 11 and 95 ± 47% determined for wild-type, Q144R, Q144R/G199D/Y223C/H329R and M213R DAAOs, respectively. The L118H DAAO mutant appears to be the best choice to quantify the entire content of D-AA since its response shows a limited dependence on the mixture composition.
Binding experiments were performed on the purified mutants in an attempt to substantiate the contribution of mutated residues to substrate/ligand recognition. Dissociation constants were measured by following the perturbation of the visible spectrum of the flavin coenzyme upon the formation of the enzymeligand complex using two classical DAAO inhibitors, i.e. benzoate and anthranilate. For all random mutants, the spectral modifications induced by benzoate and anthranilate were identical with those observed for the binding to the wild-type RgDAAO (Molla et al., 1998; Harris et al., 1999
). The binding of benzoate to L118H DAAO mutant represents the only exception: in such a case, a second spectral change at benzoate concentrations >50 mM (increase in the 420500 nm wavelength range and loss of the isosbestic points at 465 and 360 nm) was evident. Such a spectral change (Kd
280 mM) suggests that a second molecule of benzoate binds close to the isoalloxazine ring in this mutant DAAO. The Kd values summarized in Table III indicate that modest effects in binding were observed for all mutants with the ligands tested: a significantly tighter binding of benzoate (up to 3-fold) was only determined for the Q144R and Q144R/G199D/Y223C/H329R DAAO mutants. Taken together, these results indicate that the substitutions do not introduce gross perturbations of the mode of ligand/substrate binding.
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Discussion |
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Although none of the mutations introduced by error-prone PCR in the three DAAO mutants isolated in the first round of mutagenesis (Q144R, L118H and D242V/Q253R/D304V) is located in the vicinity of the active site (see below), these DAAO mutants exhibited increased catalytic efficiency on some of the substrates tested (Table II and Figure 4). Among these mutants, a 3.6-fold higher Vmax/Km ratio was determined for Q144R on D-Asp and an 2-fold higher figure for L118H on D-Arg and D-Ala in comparison with the wild-type (see Table II). The L118H mutant exhibited increased efficiency on all substrates tested. Interestingly, the residues modified in these DAAO mutants are mostly located on the protein surface (see Figure 2A). Two (Q253R and D304V) of three of the substitutions introduced in clone 2-41 belong to the wide interaction area between the two monomers (3049 Å2) (Pollegioni et al., 2002
). In particular, D304 is located on the long loop connecting ß-strands F5 and F6 (residues P302E322) and appears to play an important role in monomermonomer interaction: deletion of part of this long loop transformed RgDAAO from a dimeric protein into a stable monomer (Piubelli et al., 2002
, 2003
). In any case, its substitution is not sufficient to destabilize the monomermonomer interaction and therefore in solution this mutant is still a dimer.
Starting from the Q144R mutant, a second round of error-prone PCR was performed, still using different amplification conditions (see Table I). The two identified RgDAAO mutants show a significant change in kinetic properties with all the substrate tested (Table II). In particular, increases in Vmax,app and catalytic efficiency on D-Ala as substrate were observed for clone 5-249; this mutant also shows a higher efficiency on D-Asp and D-Arg as substrate. Among the mutations introduced in clone 5-249, two substitutions of active site residues are evident (see Figure 2B): Y223C and G199D. Y223 is involved in substrate anchoring by forming an H-bond with its -carboxylate (Umhau et al., 2000
) and has also been proposed as the group (having pKa
9.3) governing product release (Harris et al., 1999
; Pollegioni et al., 2001
). Our results indicate that the function in substrate binding exerted by Y223 can be performed even by a cysteine residue at this position, facilitating at the same time the binding of all (neutral, acidic and basic) D-AA. Concerning G199, it is 3.2 Å from the side chain of R285, the residue involved in the two-point electrostatic interaction with the substrate
-carboxylate. The introduction of an aspartic acid at position 199 should favor the interaction with the positively charged side chain of R285, optimizing the orientation of this active site residue with respect to the bound substrate. A model of the active site of the 5-249 mutant was built using the 3D coordinates of RgDAAO (Umhau et al., 2000
): a prediction of D-Asp binding in the active site of the mutant was then obtained using AutoDock 3.0 software (Goodsell and Olson, 1990
; Morris et al., 1996
; Sacchi et al., 2002
). Among the theoretical DAAOD-Asp complexes obtained for wild-type and Q144R/G199D/Y223C/H329R enzymes, the percentage of substrate molecules possessing the orientation consistent for catalysis was identical, but a significantly lower energy conformation (
G
9.5 kcal/mol) was estimated for the mutant than for wild-type DAAO, supporting the experimentally observed stabilization of D-Asp binding with the 5-249 mutant DAAO (Table II).
Recently, by a rational design approach, an RgDAAO mutant (M213R) was obtained that showed a catalytic efficiency (expressed as Vmax/Km ratio) on D-Asp and D-Glu similar to that determined for beef kidney DASPO (Sacchi et al., 2002). The appearance of appreciable activity on acidic D-AA in M213R DAAO mutant did not abolish the activity on neutral and polar D-AA, although a dramatic decrease in catalytic efficiency was observed on these substrates (an
170-fold lower Vmax/Km ratio value was determined on D-Ala) and no activity on basic D-AA was detectable using the polarographic assay (Table II) (Sacchi et al., 2002
). This new enzymatic activity is therefore not suitable for the proposed application (see also the Introduction). The substitutions introduced by the directed evolution approach modified the Vmax/Km ratios on the substrates tested to a more limited extent, but allowed the isolation of DAAO variants (e.g. L118H and T60A/Q144R/K152E) active on all D-AA (and with a remarkably improved catalytic efficiency on the acidic and the basic substrates testedwhich are poor substrates of wild-type DAAOas well as on D-Ala) and that are therefore useful for the analytical determination of the total content of D-AA in biological samples.
The substrate specificity of DAAO could be modified with both engineering approaches but, intriguingly, different structural determinants were involved. The crystal structure of an evolved aminotransferase containing up to 13 amino acid substitutions (Oue et al., 1999) showed that mutations in residues distant from the active site cause significant changes in the higher order structure of the enzyme, which influence substrate and cofactor binding. If the enzyme activity is modified, the active site must be remodeled as the result of changes in the backbone flexibility, domain motion or subunit rearrangement. Our results confirm the observation that random mutagenesis is useful in modulating substrate specificity by substitution of residue(s) far from the active site. Concerning the effect of these substitutions and in the absence of detailed structural data, it is difficult to explain the mechanism(s) by which some of these amino acid substitutions modify the substrate specificity of the evolved DAAOs. With the only exception of Y223 and G199, none of the effective amino acid substitutions lie in segments predicted to interact directly with the bound substrate. The substitutions appear to cluster on the protein surface (not far from the active site entrance): it would have been impossible to predict that these substitutions would enhance DAAO activity, apart from concluding that they synergistically generate small structural changes that affect the dynamic and/or stability of the protein in a way that enhances substrate binding or subsequently catalytic turnover. In conclusion, because of the large amount of information available on RgDAAO, its substrate specificity could be successfully modulated in a predictable and effective way by rational design (Sacchi et al., 2002
); however, an enzyme active on all D-AA could only be obtained using the directed evolution approach.
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Acknowledgments |
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Received June 1, 2004; revised July 21, 2004; accepted August 4, 2004.
Edited by Dan Tawfik
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