1Laboratory of Bioprocess Engineering, Helsinki University of Technology, PO Box 6100, 02015-HUT and 2Department of Chemistry, University of Joensuu, PO Box 111, 80101 Joensuu, Finland
3 To whom correspondence should be addressed. E-mail: ossi.turunen{at}hut.fi
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Abstract |
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Keywords: L-arabinose/crystal structure/molecular dynamics simulation/site-directed mutagenesis/xylose isomerase
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Introduction |
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Rare sugars, which are monosaccharides and their derivatives rarely found in nature, are increasingly important components for the drug industry (Ahmed, 2001; Iisakka, 2003
). For example, L-ribose is used in the preparation of antiviral agents for the treatment of hepatitis B and Epstein Barr virus (Ma et al., 1996
; Ahmed, 2001
; Iisakka, 2003
). L-Ribose is used also in the preparation of antitumor agents and siastatin B, a potent neuramidase inhibitor (Iisakka, 2003
). There are several different ways to produce L-ribose. It can be produced efficiently from L-arabinose using a molybdenum catalyst (Jumppanen et al., 2000
). L-Arabinose is abundant in nature. Mitshubishi Chemical has developed a microbial transformation process in which the starting material is D-glucose and the final product is L-ribose (Kawaguchi et al., 2001
). L-Ribose isomerase from Acinetobacter can make L-ribose from L-ribulose (Izumori and Tsusaki, 2000
), but the problem is that L-ribulose is expensive and unstable. XI can, in principle, produce L-ribose from L-arabinose, but the reaction proceeds with low efficiency.
The substrate in XI is located in a linear form between two conserved tryptophans and is liganded to metal M1, His54, Lys183 and a few Asp/Glu side chains. Lys183 forms a hydrogen bond to O1 of the substrate. The reaction mechanism involves ring opening, proton shuttling and hydride shift between C2 and C1 of the substrate (Farber et al., 1989; Collyer et al., 1990
; Whitlow et al., 1991
; Zheng et al., 1993
; Lavie et al., 1994
; Hu et al., 1997
; Asbóth and Náray-Szabó, 2000
; Garcia-Viloca et al., 2003; Fenn et al., 2004
). The ring opening appears to involve the concerted action of His54 and Asp57 as an acidbase pair (Fenn et al., 2004
). Proton shuttling including the removal of proton from O2 involves the roles of hydroxyl/H2O (located between O2 and metal M2), Asp257 and other groups. During the enzyme reaction, the metal M2 moves closer to the substrate and becomes coordinated to O1 and O2, which are then in a cis conformation. The resulting polarization of the substrate enables the hydride shift to take place. The metal M2 imposes a positive charge on the carbon C1 of the sugar and the positive charge of C1 is essential to the hydride shift (Hu et al., 1997
; Fenn et al., 2004
). Asp255 and Asp257 in Actinoplanes missouriensis interact with the metal M2, controlling its position and influencing the electrostatic properties.
We have studied earlier the reaction rates of rare sugars with XI (Pastinen et al., 1999; Vuolanto et al., 2002
). We studied subsequently, as reported now, the reasons for the low reaction efficiency of XI with L-arabinose by determining the crystal structure of Streptomyces rubiginosus XI complexed with L-arabinose. Based on this information, we modified by site-directed mutagenesis the active site of A.missouriensis XI (the mutation F26W) in order to improve the conformation of L-arabinose in the active site. Furthermore, we increased negative charge near to Asp255 and Asp257 by the mutation Q256D to study whether it is possible to affect the catalytic reaction of XI with L-arabinose by changing the key electrostatic properties close to the C1 end of the substrate. Actinoplanes missouriensis XI was used in mutagenesis studies for practical reasons, because it was produced in higher quantities in E.coli and the structures and the active sites of A.missouriensis and S.rubiginosus enzymes are highly similar [rmsd of 6XIM (Jenkins et al., 1992
) and 3XIS (Whitlow et al., 1991
) is 0.63 Å], whereas the sequence identity between these two proteins is 67%. Free energy perturbation simulations were used to study the influence of the mutation F26W on the position and conformation of L-arabinose in the active site of A.missouriensis XI.
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Materials and methods |
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Streptomyces rubiginosus XI was a gift from Genencor International (Hanko Plant, Finland). XI was co-crystallized with L-arabinose at room temperature by the hanging-drop vapor-diffusion method (McPherson, 1990). We used previously reported crystallization conditions (Carrell et al., 1984
). A droplet containing 2 ml of protein solution, 2 ml of reservoir solution and 2 ml of ligand solution was mixed and equilibrated against 0.5 ml of reservoir solution. The protein concentration was about 20 mg/ml in 25 mM maleate buffer, pH 7 (protein concentration measured by absorbance A280). The reservoir solution contained 1.5 M ammonium sulfate and 0.1 M HEPES (pH 7.5). L-Arabinose was diluted in water to a concentration of 20 mM.
For data collection, the crystals were flash-frozen in a nitrogen gas stream using 30% 2-methyl-2,4-pentanediol as cryoprotectant. X-ray diffraction data were collected at 120 K on a Rigaku RU-200HB rotating anode X-ray generator equipped with an Osmic Confocal Optics and an R-AXIS-IIC image plate detector at 1.85 Å resolution. The crystal-to-detector distance was set to 120 mm. A total of 130 frames at 1.0° oscillation were measured, exposing each frame for 10 min. The diffraction data were processed using DENZO and scaled using SCALEPACK (Otwinowski and Minor, 1997). The native S.rubiginosus XI structure, PDB code 1XIB (Carrell et al., 1994
), was used as a starting model for refinement. Refinements were carried out using CNS (Brünger et al., 1998
) and electron density map interpretation using program O (Jones et al., 1991
). Data statistics are listed in Table I.
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XI was cloned from A.missouriensis DNA by PCR into the pQE60 expression vector (Qiagen) between Nco I and HindIII sites. The mutations were introduced into the XI gene by PCR (Turunen et al., 2001). In this method, the mutations were included in the forward/reverse primer pair following the principle of Stratagene's QuikChange method. PCR was performed using Pfu Turbo polymerase (Stratagene).
Temperature-dependent activity profiles
Temperature-dependent activity profiles were measured for partially purified XI, because the knowledge of exact protein concentration is not necessary when measuring these profiles. The crude enzyme samples were produced, heat precipitated and dialyzed against the chromatography buffer as described in the section Purification of xylose isomerase (see below). Some impurities were precipitated in chromatography buffer (pH 5.8). The buffer of the samples was then changed with PD-10 columns (Sephadex G-25 M; Amersham-Pharmacia). XI was 85% pure when estimated by SDSPAGE, which also showed that the amount of impurities was equal in all samples. The temperature-dependent activity profile was determined at the pH optimum of A.missouriensis xylose isomerase in 50 mM triethanolamine buffer (pH 7.6) containing 20 mM MgSO4 and 1 mM CoCl2 (for other details, see Enzyme assays). The reaction temperatures ranged from 35 to 90°C and the reaction period at each temperature was 60 min.
Purification of xylose isomerase
Escherichia coli XL1 Blue cells were used to express A.missouriensis XI from pQE60 vector; the production was induced by 1 mM IPTG. The cells were disrupted by sonication in 0.2 M maleic acid buffer (pH 7) containing 20 mM MgSO4 and 1 mM CoCl2. After centrifugation, the E.coli proteins in the supernatant were removed by heat precipitation (1020 min, 60°C). The enzyme sample was dialyzed in chromatography buffer immediately before chromatography in order to minimize enzyme inactivation in the acidic buffer. Then XI was purified by cation-exchange chromatography (DEAE-Sepharose FastFlow; Amersham-Pharmacia). The chromatography buffer was 0.05 M sodium acetate (pH 5.8) containing 0.01 M MgCl2. The proteins were eluted from the column by a 01.0 M NaCl gradient in the chromatography buffer. XI was eluted at a salt concentration of 0.250.3 M. The buffer was changed as soon as possible by dialysis in the reaction buffer (see Enzyme assays). XI was 95% pure when estimated by SDSPAGE (Bio-Rad). The protein concentration was determined by the Bradford (1976)
protein assay using bovine serum albumin (Sigma) as standard. The enzyme samples were concentrated to 35 mg/ml with a centrifugal filter device (Millipore YM3, Amicon Bioseparations).
Enzyme assays
The reaction of XI with L-arabinose and D-glucose was measured by incubating the purified enzyme and substrate in the reaction buffer (25 mM maleic acid, pH 7, 20 mM MgSO4, 1 mM CoCl2) for various lengths of time at 60°C. The reaction was stopped with H2SO4 (final concentration 0.1 M) and the amount of sugars was measured by HPLC with an Aminex HPX-87P column (Bio-Rad). The calibration standards were D-arabinose, D-ribose, D-glucose and D-fructose. The amount of L-ribulose was determined with D-ribose as standard.
For the determination of (pseudo) Km and (pseudo) kcat, seven concentrations of L-arabinose between 83.3 and 1016 mM were used and the enzyme concentration was 2.0 g/l. The samples were incubated for 0.5, 1.0 and 2.0 h in the reaction buffer at 60°C. For the determination of Km and kcat with D-glucose, seven concentrations of D-glucose between 69.4 and 1318 mM were used and the enzyme concentration was 1.0 g/l. The samples were incubated for 5, 10 and 15 min in the reaction buffer at 60°C. The kinetic parameters were calculated using the Hyperbolic Regression Analysis function of the program Hyper that can be found at http://iubio.bio.indiana.edu:7780/archive/00000048/. The kinetic data for the reaction with L-arabinose (see Table II) were obtained for two batches of each purified enzyme. The results from two purification rounds were the same. When the kinetic parameters were determined for the conversion of L-arabinose to L-ribulose, the length of the enzyme assay was kept short so that the level of L-ribose was small enough to approximate it to zero.
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The stochastic boundary molecular dynamics simulation (Brooks and Karplus, 1983) was used to study L-arabinose modeled to the active site of the dimeric form of A. missouriensis XI (6XIM) and its mutant F26W. The dimeric subunits were chosen from the tetramer in such a way that the essential constraints for the reaction would be satisfied, namely the existence in the active site of Phe26 (Trp26 in the mutant) from the other subunit. The program CHARMM (versions 27 and 28) was used with all-atom parameter set version 22 (msi charmm force field) in the calculations (Brooks et al., 1983
; Momany and Rone, 1992
). Hydrogen atoms were built by the HBUILD facility of CHARMM (Brünger and Karplus, 1988
). The solvent molecules were built to surround protein structure and then removed if they were located outside the region of 24 Å from carbon C2 of the substrate. The water model was TIP3 (Jorgensen et al., 1983
). The reaction region was a sphere of 13 Å from carbon C2 and the buffer region was the region between 13 and 15 Å from carbon C2. A frictional coefficient of 62 ps1 was placed on all oxygens of water molecules. An average friction coefficient of 250 ps1 was used for protein atoms in the buffer region. A spherical boundary potential was used to prevent the water molecules from escaping into vacuum. TIP3P deformable boundary forces computed for a 16.8 Å sphere were used. The SHAKE algorithm (Ryckaert et al., 1977
) was used to constrain all bonds to their equilibrium values with a tolerance value of 1 x 106. A force shifting function was used to cutoff the Coulombic term at 12 Å and a van der Waals shifting function was used between 8 and 12 Å [CHARMM documentation and Steinbach and Brooks (1994)
]. A time step of 2 fs was used and the non-bonded list was updated every 20 steps based on a 13 Å list cutoff distance. The atoms in the buffer region were treated by Langevin dynamics. Simulations were performed at 333 K. Free energy curves were calculated for the rotation of C3C2C1O1 dihedral angle using free energy perturbation theory.
The same partial charges that exist for D-xylose in the modified CHARMM topology file (software CRATE version 9.0; http://comp.chem.umn.edu) were used here with minor modifications for L-arabinose. The charge value of 0.45 was usually used for the O1 atom of L-arabinose. This allowed a slightly weaker interaction with metals than the value of 0.671 in CRATE 9.0.
The charges used for D-glucose in the simulations were estimated by a combined quantum mechanicalmolecular mechanical method (Warshel and Levitt, 1976) using PM3 semi-empirical calculations (Stewart, 1989
) and Mulliken population analysis (Mulliken, 1955
) for the substrate. The substrate was handled by quantum mechanics and other groups by molecular mechanics. The calculations were performed on a stochastic boundary molecular dynamics (MD) environment. The starting structure was created using MD followed by steepest descent energy minimization. In the calculations, template-based MM charges (Momany and Rone, 1992
) for D-glucose created by Quanta were used and oxygens O2 and O4 of the substrate were constrained close to the metal M1. Finally, the structure of D-glucose was described by PM3 and it was optimized keeping O2 and O4 atoms fixed by a steepest descent algorithm using 20 000 steps. The resulting charges for D-glucose were 0.24454 (C1), 0.17716 (C2), 0.00764 (C3), 0.06765 (C4), 0.03162 (C5), 0.04393 (C6), 0.37923 (O1), 0.93150 (O2), 0.36885 (O3), 0.48322 (O4), 0.39703 (O5), 0.34248 (O6), 0.00623 (H1), 0.00874 (H2), 0.05108 (H3), 0.05819 (H4), 0.07159 (H5), 0.04650 (H6), 0.07023 (H7), 0.25673 (HO3), 0.28953 (HO4), 0.23603 (HO5) and 0.24728 (HO6).
The starting structure for the simulations with L-arabinose was created by constrained simulation keeping O2 and O4 close to the metal M1 by using the distance constraint of 2.4 Å between metals and oxygens. The distance between the metals was constrained to 4.6 Å, similar to that in the crystal structure of A.missouriensis XI (Jenkins et al., 1992). Without this constraint the metal M2 tended to move. In the final simulation, the metals were kept fixed. The distances of OD2 and OD1 atoms from the metal M2 were 2.2 and 2.2 Å for Asp255 and Asp257, respectively, resembling more our crystal structure of XI with L-arabinose than 6XIM. The distance of the N
2 atom of His220 from the metal M2 was
2.5 Å, similar to the distance in the crystal structures (6XIM and the crystal structure in this study). The charge of the side chain (CH2NH3) of Lys183 and also the charges of the metals were varied. Asp257 was in a protonated form and all histidines were uncharged with proton on ND1.
In the simulations with D-glucose, the OD2 atom of Asp255 (unprotonated) and the OD1 atom of Asp257 (protonated) were constrained to be in a more distant position from the metal M2 than in the crystal structure 6XIM. The OD2 atom of Asp255 was constrained to be 2.72.8 Å away from M2 and OD1 atom of Asp257 was constrained to be 2.93.1 Å away from M2. The N atom of Lys183 was constrained to be in an approximately similar position to that observed in the crystal structure of XI complexed with D-xylose (6XIM). Thus, the N
atom of Lys183 was constrained to be 2.82.9 Å away from the OD2 atom of Asp255. In these simulations, the O2 and O4 atoms were not fixed, but an MM charge of +2.0 was used for the metal M1 to keep O2 and O4 atoms in the vicinity of M1. The charges of +0.60 for M2 and +0.51 for the NH3 group of Lys183 were used. In contrast to the above L-arabinose simulations, we used a charge of 0.6 instead of 1.0 for the COO group of Asp292. A charge of +0.48 was used for the NH3 group of Lys294. L-Arabinose was also simulated (Figure 6C) by constraining O2 and O4 by the charge +2.0 of the metal M1 and taking the other constraints from the earlier L-arabinose simulations. Additionally, Asp257 was unprotonated and the Asp292 and Lys294 charges were the same as in the D-glucose simulation.
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Results |
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Several three-dimensional structures (native and complex) of S.rubiginosus XI have been reported earlier (Whitlow et al., 1991; Carrell et al., 1994
). The overall structure of S.rubiginosus XI is a tetramer composed of four identical monomers. Each monomer has two domains, a catalytic domain and a small domain. The catalytic domain is folded as an (
/ß)8-barrel and the small domain consists of five
-helices. The active site with two metal cations is located at the C-terminal end of the barrel. A loop from the other monomer penetrates close to the active site, with a phenylalanine residue located at the tip of the loop. The buried structure of the active site allows tight control of water molecules.
The structure of S.rubiginosus XI complexed with L-arabinose was determined at 1.85 Å resolution. The final structure has an R factor of 17.8 and an Rfree of 21.5%. The refinement statistics are listed in Table I. A clear electron density for the ligand in the active site was observed in the first map calculated with the phases derived from the protein atoms alone. In principle, the ligand in the active site could be L-arabinose, L-ribulose or L-ribose (see Figure 1). The ligand was modeled as L-arabinose because the electron density suggested that the C2 carbon is tetrahedral having a hydroxyl group in the configuration corresponding to L-arabinose. In addition, in the reaction equilibrium L-arabinose is clearly the dominant form (Vuolanto et al., 2002). The density corresponded well to the size and shape of L-arabinose (Figure 2). The interactions of L-arabinose in the active site are shown schematically in Figure 3. The crystal structure showed that L-arabinose is liganded to metal M1 by oxygens O2 and O4, to OD2 of Asp287 by oxygen O3 and oxygen O5 is hydrogen bonded to His54 (O5 is on the right end of the L-arabinose molecule in Figure 2). However, the oxygen O1 of L-arabinose (furthest left oxygen in the L-arabinose molecule in Figure 2) is in a conformation in which it is oriented away from the metal M2. In the crystal structure, the C3C2C1O1 angle is 9.0°. Based on the conformation of L-arabinose, we designed the mutation F26W that would shift the oxygen O1 closer to the metal M2. The mutation Q256D was designed to modify the electrostatic properties of the catalytically important active site residues.
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Temperature-dependent activity profiles showed that the wild-type A.missouriensis XI and the mutant F26W remained fully active for 60 min at 75°C and 80°C, respectively (Figure 4). Thus, the mutant F26W was even more stable than the wild-type enzyme. The mutant Q256D had a lower thermostability than the wild-type. It remained fully active for 60 min at 65°C, whereas the activity was lost rapidly at 80°C. Both of the mutants could be purified by heat precipitation of Escherichia coli proteins. The endogenous XI of E.coli expressed from the pQE-60 vector lost its activity after 10 min of incubation at 60°C (not shown). Hence the heat precipitation at 60°C and the performance of the enzyme assays at 60°C ruled out the possibility that the purification remnants of E.coli XI could have disturbed the enzyme assays performed with A.missouriensis XI.
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Isomerization of L-arabinose produces L-ribulose and isomerization of L-ribulose produces either L-arabinose or L-ribose (Figure 1). L-Ribose is the C2 epimer of L-arabinose. The effect of mutations on the catalytic efficiency of XI was monitored by determining the initial velocity of the reaction and the kinetic parameters at 60°C (pH 7). Both approaches showed that the mutations F26W and Q256D improved the catalytic efficiency of XI with L-arabinose (Table II and Figure 5A and B). F26W and Q256D improved the conversion of L-arabinose to both L-ribulose and L-ribose (Figure 5A and B). Hence the mutations improved the rates of both isomer production and C2 epimer production. When L-ribose was used as the substrate, no increased reaction rate was observed for the F26W mutant (not shown). Hence the effect of F26W was specific to L-arabinose. In contrast, the mutation Q256D increased the reaction rate (initial velocity) of XI about 6-fold with L-ribose (not shown). The effect of the mutations was not caused by a shift in the pH optimum, since the pH optimum was approximately the same for the wild-type enzyme and the mutants (pH 7.58.5; data not shown).
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The determination of kinetic parameters for the conversion of L-arabinose to L-ribose was not attempted because the reaction goes through an intermediate, L-ribulose, which in turn is converted to L-ribose and back to L-arabinose.
Reaction with D-glucose
The enzymatic properties of the mutants were studied with D-glucose to assess the effect of the mutations on the normal functioning of the enzyme. The kinetic parameters were determined for the conversion of D-glucose to D-fructose with the wild-type enzyme and the mutant F26W (Table III). The reaction efficiency of the mutant F26W with D-glucose was similar to that of the wild-type enzyme. This indicated that the bulky Trp side chain at position 26 did not significantly disturb the positioning of D-glucose in the active site. The reaction efficiency of the mutant Q256D for the conversion of D-glucose to D-fructose was also similar to that of the wild-type enzyme (not shown).
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The free energy perturbation calculations were performed with A.missouriensis XI. A free energy plot was created around the dihedral angle C3C2C1O1 using stochastic boundary molecular dynamics. The simulations proposed three conformational minimum positions for the L-arabinose C3C2C1O1 dihedral angle, which were 0 to 10°, 120 to 140° and +130 to +150° (Figure 6B). The third minimum (+130 to +150°) appeared to be occupied by a very small structural population. D-Xylose has values between 155 and 170° for the C3C2C1O1 dihedral angle in the crystal structure (6XIM). In this conformation, O1 is hydrogen bonded to Lys183. In general, the position of O1 of the substrate depends on the strength of the interactions from the metal M2 and Lys183. During the hydride shift, when the metal M2 is liganded to the substrate, oxygens O2 and O1 are in a cis conformation, meaning that the dihedral angle C3C2C1O1 is 120° and O2C2C1O1 is close to zero (Whitlow et al., 1991; Hu et al., 1996
, 1997
; Garcia-Viloca et al., 2003
).
Different combinations of charges were used for the metals M1 and M2 (Figure 6A). In particular, the depth of the energy minimum at 0 to 10° was sensitive to the changes in the charge values of the metals (Figure 6A). The charge of the Lys183 side chain (CH2NH3) was also varied. A higher positive charge of Lys183 m and higher charges of metals changed the conformational equilibrium in L-arabinose from the conformation at about 9° (crystal structure) towards conformation at 120° of the C3C2C1O1 dihedral angle (Figure 6A). For example, when the charges were +0.8 for M1, +0.5 for M2 and +0.39 for Lys183, we obtained a result that the population of the substrate in a conformation having the dihedral angle C3C2C1O1 close to 120° was less than 50% (Figure 6A). If the charges of metals were set to higher values, then the charge of Lys183 had to be significantly lowered in order to obtain a global free energy minimum at a dihedral angle of 0 to 10° (Figure 6A).
In the simulations comparing the wild-type XI and the mutant F26W, both complexed with L-arabinose, the charge values were +0.8 for M1, +0.5 for M2 and +0.39 for Lys183 (Figure 6B). Whereas these charge values were slightly lower than those that have been calculated by using the methods of quantum chemistry for S.rubiginosus XI complexed with D-xylose (Hu et al., 1997), the results obtained with them were consistent with the conformation of L-arabinose in the crystal structure. The simulation of L-arabinose in the modeled structure of the F26W mutant XI showed that the mutation slightly disfavored the conformation at a dihedral angle of 0 to 10°, in which the oxygen O1 was turned away from the metal M2 and Lys183 (Figure 6B).
To compare L-arabinose and D-glucose, they were both simulated by constraining the substrate to stay in the vicinity of the metal M1 by using a charge of +2.0 for M1. The simulation results for L-arabinose were largely similar between this and the approach described above. However, to obtain a similar distribution of the conformations (Figure 6) in the second approach (+2.0 charge of M1) than the earlier one (fixing of O2 and O4), slightly higher charges of the metal M2 and particularly the NH3 group of Lys183 were required. The charge values were +0.60 for the metal M2 and +0.51 for the NH3 group of Lys183. In the simulation of XI with D-glucose, when the C3C2C1O1 dihedral angle was constrained to be close to 0°, the average distance between O1 atom and N atom of Lys183 was
4.9 Å, whereas the corresponding distance was 5.4 Å in the L-arabinose simulation and 5.8 Å in the crystal structure of XI with L-arabinose. This result also indicates that there is a difference in position between D-glucose and L-arabinose in the active site of XI.
The free energy profile for D-glucose had three potential energy minima (Figure 6C), whereas the populations of different conformations differed clearly from those of L-arabinose. In D-glucose, the most populated conformation had the dihedral angle C3C2C1O1 of +140°. In this conformation O1 is hydrogen bonded to the NH3 group of Lys183. Apparently, the interactions from the metal M2 and Lys183 keep O1 of D-glucose away from the conformation detected for L-arabinose in the crystal structure.
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Discussion |
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The crystal structure of XI complexed with L-arabinose is reported here for the first time. The structure revealed problems that apparently cause the low reaction rate of XI with L-arabinose. The S.rubiginosus XI structure complexed with L-arabinose was superimposed with the S.rubiginosus XI structure complexed with D-xylose, determined at 1.6 Å resolution (3XIS) (Whitlow, et al., 1991) (Figure 7). The comparison showed that the conformations of active site residues were similar. In addition, the binding mode of L-arabinose to metal M1 was similar to that of D-xylose. The difference between D-xylose and L-arabinose is in the stereochemistry of the carbon C4. The structural comparison indicates that the hydroxyl groups at O4 and O5 bind similarly to the active site despite the different stereochemistry at C4. However, the position of L-arabinose is shifted about 0.8 Å away from the site observed for D-xylose. Thus, the oxygen O2, which is modified in the reaction, is 3.7 Å away from the catalytic metal M2 in the D-xylose complex, whereas it is 4.4 Å away in the L-arabinose complex. In addition, the conformation around the C1C2 bond was changed. The torsion angle for the C3C2C1O1 bond was 9°, whereas it is +179° for D-xylose in S.rubiginosus XI (3XIS; Whitlow, et al., 1991
) and 153 to 161° in A.missouriensis XI (6XIM; Jenkins et al., 1992
). The distance from the reactive O1 atom of L-arabinose to the metal M2 is 5.5 Å and to N
of Lys183 it is 5.9 Å. The corresponding values for D-xylose in 3XIS are 3.5 and 3.2 Å, respectively, and thus significantly shorter. Hence the reaction of XI with L-arabinose is affected by the orientation of O1 away from the metal M2 and Lys183. The hydrogen bond between Lys183 and the oxygen O1 of the substrate has been proposed to have a role in holding the substrate in the optimal position for the reaction (Lambeir et al., 1992
; Whitaker et al., 1995
; Hu et al., 1996
; Fenn et al., 2004
). The long distance of O1 from N
of Lys183 (5.9 Å) prevents the formation of a hydrogen bond. These findings explain the low reaction rate with L-arabinose, which is over 100 times lower than that with D-glucose (Vuolanto et al., 2002
).
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The hydrogen bond between O1 and Lys183 has been assumed to stabilize the open substrate conformation (Lambeir et al., 1993; Asbóth and Náray-Szabo, 2000; Fenn et al., 2004
). It is therefore important to note that despite the lack of the hydrogen bond between O1 and Lys183, L-arabinose still stayed in an open conformation in the active site. It therefore appears that this hydrogen bond is important for correct positioning of O1, enabling the metal M2 to coordinate to O1.
The MD simulations indicated that L-arabinose and D-glucose have three energy minima for the C3C2C1O1 dihedral angle. L-Arabinose had a very small population in the energy minimum, in which the oxygen O1 is hydrogen bonded to Lys183, whereas this conformation was the most populated one in D-glucose. Although giving information about interactions of the substrate in the active site, the depths of the energy minima were not simulated accurately enough to predict exactly the position of oxygen O1 in the active site. However, even in the crystal structures the conformation of O1 may deviate between the different monomers in the tetrameric structure (e.g. O1 of D-xylose in 6XIM).
Mutation F26W
Phe26 penetrating into the active site from another subunit of XI is located near the O1 of L-arabinose (Figure 8). The mutation F26W was designed to shift O1 away from its wrong position revealed by the crystal structure of S.rubiginosus XI with L-arabinose. The mutation F26W was done for practical reasons in A.missouriensis XI, because the effect was expected to be similar in both enzymes. F26W improved to some extent the reaction of XI with L-arabinose. The improvement of the reaction rate with L-arabinose was mainly due to the increase in kcat. This indicated that the mutation had not necessarily altered the binding affinity of the substrate to the active site, but instead, the catalytic reaction had become faster. An important factor in the reaction is the coordination of carbonyl oxygen O1 of the substrate. When the metal-catalyzed hydride shift occurs during the enzyme reaction between carbons C2 and C1, the oxygen O1 has to be in a cis conformation with O2 (the dihedral angle of C3C2C1O1 is about 120° and that of O2C2C1O1 is about 0°) (Whitlow et al., 1991). The molecular dynamics simulations of L-arabinose in the active site of the A.missouriensis XI indicated that there are three main conformations for the substrate. The simulations of the F26W mutant complexed with L-arabinose suggested that the cis conformation of O1 and O2 (the C3C2C1O1 angle is 120° to 130°) becomes more populated in the mutant than in the wild-type enzyme.
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Mutation Q256D
We also introduced other mutations into the vicinity of the active site. The mutation Q256D was expected to change the electrostatic field near the substrate and near the residues important for the reaction mechanism, although the side chain of the residue 256 is about 10 Å away from the catalytic metal M2 (Figure 8). Residue 256 is located in the polypeptide chain between two aspartates, Asp255 and Asp257, which bind to the catalytic metal M2 and participate in the proton shuttling and polarization of the substrate. The side chain of Gln256 points towards the opposite direction where it forms a hydrogen bond with the main chain nitrogen of Asn250 (in S.rubiginosus) or His250 (in A.missouriensis).
Q256D improved the reaction rate of XI with L-arabinose. The improvement in the reaction rate was due to the improvement of both kcat and Km and the increase in the kcat/Km ratio was 2.8-fold. The preliminary calculations of pKa values with the modeling program WHATIF (without ligands in the active site) indicated that Q256D changes the pKa values of residues Asp255, Asp257 and His220 (data not shown). It could be that the mutation affected the charge and even the conformation of the catalytically important side chains and metal M2.
In conclusion, we have shown in this study that the reaction efficiency of XI with a weakly reacting substrate such as L-arabinose can be modified based on structural information. XI could thus be tailored to perform new kinds of reactions, which might be chemically and industrially significant. We were able to use MD simulations to model the effect of the mutation F26W on the reaction of XI with L-arabinose. Furthermore, the differences between the interactions of L-arabinose and D-glucose in the active site were modeled by MD simulations. The site-directed mutagenesis together with X-ray diffraction and simulation studies with weak substrates are useful for obtaining an understanding of the nature and functioning of the very complex active site of xylose isomerase.
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Received July 2, 2004; revised January 4, 2005; accepted January 4, 2005.
Edited by Bauke Dijkstra