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Structure determinants of substrate specificity of hydroxynitrile lyase from Manihot esculenta

¹ Institut für Organische Chemie der Universität Stuttgart, D-70569 Stuttgart, Germany
² Institut für Zellbiologie und Immunologie der Universität Stuttgart, D-70569 Stuttgart, Germany

Reprint requests to: Hanspeter Lauble, Institut für Organische Chemie, Universität Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart, Germany; e-mail: PeterLauble{at}t-online.de; fax: 49-711-685-4269.

(RECEIVED August 9, 2001; FINAL REVISION September 25, 2001; ACCEPTED October 8, 2001)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/ps.33702.

	Abstract

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Tryptophan 128 of hydroxynitrile lyase of Manihot esculenta (MeHNL) covers a significant part of a hydrophobic channel that gives access to the active site of the enzyme. This residue was therefore substituted in the mutant MeHNL-W128A by alanine to study its importance for the substrate specificity of the enzyme. Wild-type MeHNL and MeHNL-W128A showed comparable activity on the natural substrate acetone cyanohydrin (53 and 40 U/mg, respectively). However, the specific activities of MeHNL-W128A for the unnatural substrates mandelonitrile and 4-hydroxymandelonitrile are increased 9-fold and 450-fold, respectively, compared with the wild-type MeHNL. The crystal structure of the MeHNL-W128A substrate-free form at 2.1 Å resolution indicates that the W128A substitution has significantly enlarged the active-site channel entrance, and thereby explains the observed changes in substrate specificity for bulky substrates. Surprisingly, the MeHNL-W128A–4-hydroxybenzaldehyde complex structure at 2.1 Å resolution shows the presence of two hydroxybenzaldehyde molecules in a sandwich type arrangement in the active site with an additional hydrogen bridge to the reacting center.

Keywords: Substrate specificity; active-site tunnel mutant; crystal structure

	Introduction

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Hydroxynitrile lyases (HNLs) constitute a diverse family of enzymes that catalyze the stereospecific cleavage of a wide range of cyanohydrins into aldehydes or ketones and hydrogen cyanide (cyanogenesis) (Wajant and Effenberger 1996), which have recently become the subject of intensive structural studies (Wagner et al. 1996; Zuegg et al. 1999; Lauble et al. 2001a,b). Apart from the interesting aspects of their catalytic function, HNLs are of practical importance as biocatalysts for the reverse reaction of cyanogenesis, that is, the stereoselective addition of HCN to carbonyl compounds (Scheme 1).

View larger version (14K): [in this window] [in a new window]   Scheme 1.

On the basis of the X-ray structure of MeHNL (Lauble et al. 1999), first attempts have been undertaken to correlate the substrate specificity with structural features of the three-dimensional structure of the enzyme. The X-ray structure of wild-type MeHNL revealed that the active site of the enzyme is only accessible via a narrow tunnel in the surface of the protein (Lauble et al. 2001a). The tunnel entrance is capped by Trp 128, which, therefore, might be a primary determinant for substrate transport into the active site. To investigate the functional importance of Trp 128 for substrate specificity, this residue was substituted by alanine. Whereas the catalytic activity of this mutant toward acetone cyanohydrin was almost unchanged, the conversion of mandelonitrile and 4-hydroxymandelonitrile was significantly increased. To address this change in substrate specificity of the mutant enzyme, we have determined the X-ray crystal structure of MeHNL-W128A in complex with 4-hydroxybenzaldehyde at 2.1 Å resolution. Additionally, the three-dimensional structure of MeHNL-W128A was determined at 2.1 Å resolution in a substrate-free form to characterize any structural alterations that might have resulted from the mutation.

	Results and Discussion

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Kinetical characterization and X-ray structure of MeHNL-W128A
Wild-type MeHNL and MeHNL-W128A were expressed in Escherichia coli and purified by anion exchange chromatography to homogeneity (>95%) (Wajant et al. 1996). The purified enzymes were characterized with respect to their enzymatic activities by use of acetone cyanohydrin, mandelonitrile, and 4-hydroxymandelonitrile as substrates.

The MeHNL-W128A mutant shows a 30% reduced relative specific activity for the natural substrate acetone cyanohydrin compared with the wild-type enzyme (Table 1). However, MeHNL-W128A is much more potent in cleavage of mandelo- and 4-hydroxymandelonitrile than wild-type MeHNL. Thus, MeHNL-W128A mutant exerts a 9-fold increase in specific activity for mandelonitrile and a nearly 450-fold increase in specific activity for 4-hydroxymandelonitrile. Hence, Trp 128 of MeHNL has a significant impact on enzyme activity for bulky substrates. In accordance with the specific activities for the three substrates (Table 1), the K_m value of MeHNL-W128A for acetone cyanohydrin is slightly higher than that for the wild-type enzyme, whereas the K_m values for the sterically demanding substrate mandelonitrile is seven times lower (Table 1). For 4-hydroxymandelonitrile, a K_m value of wild-type MeHNL could not be determined. Due to the slow conversion rate of the substrate, the enzyme activity was below the detection limit at low substrate concentrations.

View larger version (51K): [in this window] [in a new window]   Fig. 1. (A) Molecular surface drawing of wild-type MeHNL (left) and MeHNL-W128A-4–hydroxybenzaldehyde complex (right). The enzymes are viewed toward the active-site tunnel entrance. The surface of Trp 128 (left) and Ala 128 (right) is colored yellow, showing the completely covered entrance of the wild-type enzyme and the wide-open substrate binding site of the mutant enzyme. Stereoview of the MeHNL-W128A active site in complex with 4-hydroxybenzaldehyde. (B) |Fo|-|Fc| electron density omit map contoured at 4. (C) Intermolecular interactions of the substrate molecules and selected active-site residues. Hydrogen bonds are shown as red broken lines, van der Waals interactions as black broken lines.

X-ray structure of MeHNL-W128A in complex with 4-hydroxybenzaldehyde
The crystal structure of MeHNL-W128A mutant enzyme in complex with 4-hydroxybenzaldehyde was determined at 2.1 Å resolution and refined with R = 19.7% and R_free = 24.4% (Table 2). In the active site of molecules A and B, the electron density for 4-hydroxybenzaldehyde is well resolved, clearly indicating the orientation of the molecule (Fig. 1B). The carbonyl oxygen forms hydrogen bonds to Ser 80-OG (2.3 Å), Thr 11-OG (2.6 Å), and to the active-site water X122 (2.8 Å). The phenyl ring shows van der Waals contacts with four tunnel residues including C2 with Ile 12-CG1 (3.7 Å) and Leu 149-CD2 (3.8 Å), C3 with Ile 12-CD1 (3.7 Å), Met 147-SD (3.7 Å) and Leu 149-CD2 (3.8 Å), and C6 with Cys81-SG (3.7 Å). The hydroxyl group forms no hydrogen bonds with protein residues. The binding of a second molecule 4-hydroxybenzaldehyde into the active site of molecules A and B is a most surprising result. The 2|F_o|-|F_c| electron density map (Fig. 1B) clearly shows the position and orientation of the second molecule, that could be modeled in such a way that its hydroxyl group is stabilized at the base of the active-site cavity forming a hydrogen bond to the active-site water X122 (2.8 Å). The phenyl ring of the second molecule makes van der Waals contacts to three tunnel residues. Most specifically, C2 interacts with Ile 210-CG1 (3.4 Å), C3 with Ile 210-CD1 (3.9 Å), C4 with Leu 149-CD2 (4.0 Å) and Leu 153-CD2 (4.0 Å), and C5 with Leu 149-CD2 (3.8 Å) and Ile 210-CD1 (4.0 Å). The carbonyl group points toward the tunnel entrance and interacts with the main-chain oxygen of Phe 125 (3.1 Å). These interactions place the phenyl ring of the second molecule in a sandwich type mode to the first one, thereby allowing aromatic stacking interactions. The distance between the two aromatic planes is 3.8 Å. The two molecules bind in a head-to-tail arrangement and are bridged by the active-site water molecule, which is also hydrogen bonded to the catalytic residues His 236-NE2 (2.7 Å) and Ser 80-OG (3.0 Å).

Figure 1C represents schematically the binding of 4-hydroxybenzaldehyde to the active site of MeHNL-W128A mutant enzyme, including hydrogen bonding and van der Waals interactions of the two molecules and the protein. The average B factor for the productive mode bound hydroxybenzaldehyde in the active site of molecules A and B is 41 Ų, whereas the average B factor for the second hydroxybenzaldehyde is 53 Ų (Table 2). The higher B factor for the latter probably reflects an occupancy of <1.0, which was the value for occupancy used in the refinement.

Superposition of the MeHNL-W128A–hydroxybenzaldehyde structure onto the MeHNL-W128A substrate-free structure shows an rms deviation of 0.5 Å for all atoms. In the complexed MeHNL-W128A structure, the carbonyl oxygen O1 is hydrogen bonded to the active-site water X122, replacing the MPD 4-hydroxyl group interaction observed in the substrate-free form. The active-site water X122 is hydrogen bonded to His 236-NE, as observed in the substrate-free complex, and additionally to the 4-hydroxyl group of the second hydroxybenzaldehyde molecule. Although the position of water X122 does not seem appreciably altered by the binding of two carbonyl substrates, Ser 80 undergoes a conformational change upon hydroxybenzaldehyde binding. Ser 80 reorients in a manner making a hydrogen bond between Ser 80-OG and His 236-NE, thereby favoring the abstraction of a proton by the general base His 236 as required in the catalytic mechanism (Lauble et al. 2001a,b). In addition, Ser 80-OG makes an unusually short hydrogen bond to the carbonyl oxygen O1 (2.3 Å). As a consequence of the Ser 80 reorientation, the hydrogen bond of OG to the substrate-free active-site water is lost and the hydrogen bond between the hydroxyl group of Ser 80 and Thr 11 lengthens from 3.0 to 3.6 Å, thereby allowing a new interaction of Thr 11-OG to the carbonyl oxygen of hydroxybenzaldehyde, increasing the total number of possible hydrogen bonds at the active site by two compared with the substrate-free complex.

Superposition of the MeHNL-W128A–hydroxybenzaldehyde complex onto wild-type MeHNL–acetone structure (Lauble et al. 2001a) shows an rms deviation of 0.2 Å for all backbone atoms and an rms deviation of 0.2 Å for active-site residues. The comparison shows that hydroxybenzaldehyde binds to the MeHNL-W128A active site, as does acetone, by interaction of the carbonyl oxygen with Ser 80-OG and Thr 11-OG, respectively. A surprising difference of the hydroxybenzaldehyde complex is the 90° rotation of the carbonyl plane with respect to that of acetone. The difference in the orientation means that the phenyl ring of 4-hydroxybenzaldehyde points perpendicular toward the tunnel entrance, whereas the corresponding methyl group in acetone lies more parallel to the entrance. The carbonyl oxygen, however, maintains basically the same position seen for the carbonyl oxygen in the MeHNL–acetone complex. The comparison also shows that the carbonyl oxygen of hydroxybenzaldehyde is additionally hydrogen bonded to the active-site water X122, a water molecule not observed in the wild-type MeHNL–acetone complex. The active-site water is also present in the MeHNL-W128A substrate-free form, in MeHNL-S80A substrate-free form (Lauble et al. 2001b), and in the wild-type MeHNL–chloroacetone structure (Lauble et al. 2001a).

In conclusion, the MeHNL-W128A–hydroxybenzaldehyde complex reveals a largely open-ended S2 pocket, thereby facilitating the accommodation of larger hydrophobic substrates most significantly demonstrated by the presence of two hydroxybenzaldehyde molecules in the active site.

The facilitated access to the active site of the MeHNL-W128A mutant enzyme compared with the wild-type enzyme could be proved. From specific activity and K_m value, a negligible steric influence could be deduced for the natural substrate acetone cyanohydrin. On the contrary, a reduced steric hindrance in the mutant MeHNL-W128A seems to cause clearly improved K_m values and increased specific activities for the bulkier substrates mandelo- and 4-hydroxymandelonitrile. Superposition of the MeHNL-W128A–hydroxybenzaldehyde complex onto the wild-type MeHNL–acetone crystal structure (Lauble et al. 2001a) shows that binding of hydroxybenzaldehyde into the wild-type MeHNL active site would result in a steric clash of the 4-hydroxyl group with the indole side chain of Trp 128. The substrate preference for the mutant enzyme, however, is not exclusively determined by size exclusion. Because the specific activity, as well as the K_m value, for 4-hydroxymandelonitrile are clearly improved in comparison with mandelonitrile, the defined arrangement of a second molecule 4-hydroxybenzaldehyde in the active site of MeHNL-W128A seems to have an additional influence on the catalytic mechanism. From the experimental results, however, it is difficult to estimate the influence of the additional hydrogen bridge or the aromatic -stacking on the unusually high substrate specificity for 4-hydroxymandelonitrile.

	Materials and methods

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Expression and purification of MeHNL-W128A
E. coli XL1-Blue (Stratagene) was used for the propagation of plasmids. Site-directed mutagenesis of MeHNL were achieved with the pQE4-MeHNLwild-type expression plasmid (Wajant and Pfizenmaier 1996). The W128A mutation was introduced into pQE4-MeHNLwild type using the Quick Change Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's recommendations with two complementary primers corresponding to nucleotides 383–435 of MeHNL (5`-AAGCTTTTGGAGT CGTTTCCTGCGAGAGACACAGAGTATTTTACGTTCAC-3`) containing the mutation of interest (bold underlined). Wild-type MeHNL and MeHNL-W128A were expressed in an E. coli strain M15[pREP4] as described previously (Wajant et al. 1996). Purification of the recombinant proteins were achieved by use of anion exchange chromatography and gel exclusion chromatography (Wajant et al. 1996).

Enzyme assays and enzyme kinetics
Cleavage of acetone cyanohydrin by wild-type MeHNL and MeHNL-W128A was measured as described by Selmar et al. (1987). For acetone cyanohydrin, the amount of HNL that decomposes 1 µmole of the substrate in 1 min at room temperature (pH 5.2) in 100 mM sodium acetate is defined as 1 unit. MeHNL-catalyzed cleavage of mandelonitrile was determined according to Hanefeld et al. (1999), whereas the cleavage of 4-hydroxymandelonitrile was determined as described by Seely et al. (1966). Total protein for calculations of specific activities was determined with the BCA Protein Assay Reagent (Pierce) according to the manufacturer's recommendations.

For determination of the K_m-values for wild-type MeHNL and MeHNL-W128A mutant enzyme for acetone cyanohydrin, mandelo- and 4-hydroxymandelonitrile, the initial velocity was determined over a substrate concentration of 1–55 mM for acetone cyanohydrin, 1–17 mM for mandelonitrile, and 1–1000 µM for 4-hydroxymandelonitrile, respectively, in sodium acetate (pH 5.2) at room temperature. The obtained data were analyzed according to the method of Michaelis and Menten (Palmer 1981). Accurate determination of v_max values were obtained from Lineweaver-Burk plots (Palmer 1981).

Crystallization and data collection
For crystallization, the protein was dialyzed against 10 mM sodium citrate (pH 5.4) and concentrated to 32 mg/ml. Crystals of MeHNL-W128A mutant enzyme were grown by the vapor-diffusion hanging-drop method under the conditions described previously (Lauble et al. 1999) and belong to the tetragonal space group P4₁2₁2 (Table 2), with two molecules per asymmetric unit and are isomorphous to wild-type MeHNL. For data collection of the MeHNL-W128A substrate-free form, a single crystal was transferred into a stabilization buffer containing 100 mM sodium citrate (pH 5.4), 5% PEG 8000, 28% MPD, and flash cooled at 100 K.

MeHNL-W128A–hydroxybenzaldehyde complex crystals were prepared by soaking a MeHNL-W128A crystal in 100 mM sodium citrate (pH 5.4), 8% PEG 8000, and 28% MPD containing 400 mM 4-hydroxybenzaldehyde for 1 h. The soaked crystal was flash cooled at 100 K. Diffraction data were collected on a 30-cm MAR research image plate using the synchrotron radiation at station PX11 ( = 0.902 Å) at EMBL outstation, Hamburg, Germany. The exposing dose was 10,000 per frame using 1° oscillation. The crystal-to-detector distance was 200 mm, and the slits were set at 0.2 mm. MeHNL-W128A mutant crystal diffracted to 2.1 Å resolution. Mosaicity of the frozen crystals was in the range of 0.2°. Raw data images were indexed and integrated using the program DENZO (Otwinowski 1993), and all data scaled and reduced with the programs from the CCP4 suite (Collaborative Computational Project, Number 4 1994). The final statistics on the data set are given in Table 2.

Refinement
The starting model for the refinement of both structures was the coordination set of the refined structure of the MeHNL–acetone complex (PDB entry 1DWP) (Lauble et al. 2001a) without water molecules and ligands. This crystal structure contains two protein molecules in the asymmetric unit (A and B) and consists of 262 amino acids for molecule A and 258 amino acids for molecule B. The extra four amino-terminal residues observed for molecule A are part of the seven residues encoded by the multiple cloning site of the expression vector used for the overexpression of MeHNL (Wajant and Pfizenmaier 1996). The Trp 128 residue was substituted by alanine. To account for the pronounced anisotropic diffraction properties of MeHNL crystals (Lauble et al. 1999), an anisotropic correction was included in the refinement. The electron density maps were displayed using the graphic program XTALVIEW (McRee 1999), and the atomic model for hydroxybenzaldehyde and MPD were generated and geometrically minimized with INSIGHT II/DISCOVER (BIOSYM/ Molecular Simulations Inc., Release 95.0, 1995). Each structure was refined using the same protocol, starting with a rigid-body refinement to allow rearrangement of molecules A and B in the asymmetric unit against each other. Subsequent rounds of positional refinement proceeded in steps, each step including data shells of increasing resolution. When the resolution was extended to 2.5 Å, 2|F_o| – |F_c| electron density maps were calculated and each protein model analyzed. The engineered W128A mutation was clearly defined, and water molecules were added using the MeHNL–acetone complex structure as a guideline. Each model was subjected to further rounds of positional, simulated annealing (T = 3000 K) and B-factor refinement including, in the last step, all data in the resolution range 8–2.2 Å. For each structure, 2|F_o|–|F_c| and |F_o|–|F_c| electron density maps were calculated to check the water molecules. Water molecules with B factors better than 60 Ų were retained. The electron density map for the MeHNL-W128A substrate-free form shows one well-resolved water molecule in the active site of molecule A, two in the active site of B, and additionally continuous electron density that could clearly be attributed to the crystallization compound MPD. Analysis of the 2|F_o|–|F_c| map calculated for MeHNL-W128A–hydroxybenzaldehyde shows clear electron density for the aldehyde in the active site of molecules A and B. Electron density is also observed for an active-site water molecule in both subsites. An additional extraneous electron density in the active site of both molecules A and B, showing basically the same long plane shape as hydroxybenzaldehyde, was observed and therefore modeled as 4-hydroxybenzaldehyde. Both MeHNL-W128A mutant structures along with the water molecules were refined against their diffraction data between 8–2.1 Å resolution. 2|F_o|–|F_c| maps were calculated to confirm the fit of these models to the active-site density. The refinement of the MeHNL-W128A substrate-free structure and the MeHNL-W128A–hydroxybenzaldehyde complex structure converged to R = 19.2%, R_free = 23.4% and R = 19.7%, R_free = 24.4%, respectively (Table 2). The geometric parameters of both models were analyzed with PROCHECK (Laskowski et al. 1993) and are within the expected deviation (Table 2). A Ramachandran plot shows that the residues Ser 80 and Arg 129 of each subunit fell outside of the energetically favorable regions. Ser 80 is located in the active site and shown clearly in the electron density map, whereas Arg 129 deviates from ideal values because of strain from a salt bridge to Glu 156. The two molecules in the asymmetric unit are very similar for both structures and superimpose with rms differences of 0.7 Å for all atoms. Molecule A of each structure is used for analysis in this paper. The final coordinates of the MeHNL-W128A structures have been deposited with the Protein Data Bank (PDB entry 1EB8 for MeHNL-W128A, PDB entry 1EB9 for MeHNL-W128A–hydroxybenzaldehyde complex).

	Acknowledgments

 
We thank the EMBL outstation at DESY, Hamburg, for use of data-collection facilities and beam line X11, and Dr. A. Baro for her help in preparing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft, Grant Wa I025/1–1 + 1–2 and by the Bundesministerium für Bildung und Forschung, Grant B3.8U – Zentrales Schwerpunktprogramm Bioverfahrenstechnik Stuttgart.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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