EPR Study of Substrate Binding to Mn(II) in Hydroxynitrile Lyase from

Jun 22, 2016 - On the Lewis acidic vacant coordination site the Mn binds either substrate or the product, leading to a hexacoordinated 17 electron ...
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EPR Study of Substrate Binding to Mn(II) in Hydroxynitrile Lyase from Granulicella tundricola Femke Vertregt,† Guzman Torrelo,† Sarah Trunk,§ Helmar Wiltsche,‡ Wilfred R. Hagen,† Ulf Hanefeld,*,† and Kerstin Steiner*,§ †

Biokatalyse, Afdeling Biotechnologie, Technische Universiteit Delft, van der Maasweg 9, 2629HZ Delft, The Netherlands Institute of Analytical Chemistry and Food Chemistry, TU Graz, Stremayrgasse 9/III, 8010 Graz, Austria § Austrian Centre of Industrial Biotechnology GmbH, Petersgasse 14/4, 8010 Graz, Austria ‡

S Supporting Information *

ABSTRACT: GtHNL from Granulicella tundricola is a Mn(II) containing hydroxynitrile lyase with a cupin fold. The quasi-octahedral manganese is pentacoordinated by the enzyme. It catalyzes the enantioselective addition of HCN to aldehydes, yielding R-cyanohydrins. On the Lewis acidic vacant coordination site the Mn binds either substrate or the product, leading to a hexacoordinated 17 electron complex. EPR spectra of the active enzyme are unusually wide with a zero-field splitting approximately equal to the X-band microwave energy. A spectral change is induced by incubation with either one of the substrates/products HCN, benzaldehyde, and/or mandelonitrile. This points toward Mn(II) catalyzed cyanohydrin synthesis.

KEYWORDS: hydroxynitrile lyase, cupin, Mn, Lewis acid catalysis, cyanohydrin, EPR

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INTRODUCTION The bacterial hydroxynitrile lyase (HNL) from Granulicella tundricola (GtHNL) belongs to the cupin superfamily of proteins and is metal dependent.1 The pentacoordinated manganese is surrounded by four histidines and one glutamine; in the crystal structure the vacant position of the octahedral metal is occupied by water (Figure 1).1 In contrast to other metal-binding proteins with cupin fold, in which two to four metal binding amino acids are reported,2 GtHNL has a fifth amino acid that can act as a ligand: histidine 96. The amino acid exchange of H96 to alanine results in inactive enzyme, while no change in metal loading is observed. When the other three amino acids that coordinate in the square plane of the octahedron are exchanged (H53A, H55A, and Q59A), metal and activity loss is observed, while H94A has little influence on metal binding and activity.1 Close examination of the structure reveals that the metal is centered but below the plane of the four square planar ligands and that in particular the water molecule is dislocated relative to an ideal octahedron (Figure 1A-C and Tables 1 and 2). More recently a variant with significantly increased activity was described, GtHNL-A40H/ V42T/Q110H.1b The metal is likewise octahedrally coordinated with water as the sixth ligand (Figure 1D,E) and with a very similar geometry. Again manganese is slightly below the plane of ligands H53, H55, Q59, and H96, and the water molecule is skewed, though less pronounced than in GtHNL (Tables 1 and 2). GtHNL catalyzes the R-selective formation and decomposition of cyanohydrins. While the wild type has modest activity, GtHNL-A40H/V42T/Q110H has a greatly improved © XXXX American Chemical Society

activity and in fact catalyzes the conversion of sterically demanding o-chlorobenzaldehyde with excellent enantioselectivity.1b The catalytic activity of GtHNL is tightly connected to the pentacoordinating cupin structure. Even in GtHNL-A40H/ V42T/Q110H none of the altered amino acids acts as ligand of manganese, indicative for the importance of the amino acids H53, H55, Q59, H94, and H96 as ligands of manganese for its catalytic activity. Similarly coordinated Mn(III)-salen complexes are successfully utilized for asymmetric cyanohydrin synthesis, while correspondently coordinated Mn(II) is catalytically inactive.3 These homogeneous Mn(III) catalysts work with a combined Lewis acid and Lewis base catalysis, the Mn(III) acting as Lewis acid while a basic group attached to the ligand functions as Lewis base.3 This would suggest manganese to have oxidation state Mn(III) in GtHNL and work via a similar manner, H106 acting as Lewis base. This is supported by the increased activity of GtHNL-A40H/V42T/Q110H that has two additional histidines in the active site; they too can act as Lewis bases. The nature of the central manganese ion therefore needs to be explored. For this investigation the wild type GtHNL, GtHNL-H96A, and GtHNL-A40H/V42T/Q110H are studied. Received: April 28, 2016 Revised: June 20, 2016

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Table 1. Distances of Ligands to Mn and of Octahedral Corner Points for GtHNL and GtHNL-A40H/V42T/Q110H from H2O

Mn

HIS 94

HIS 53 HIS 55 GLN 59 HIS 96

to

GtHNL (Å)

GtHNL-A40H/V42T/Q110H (Å)

HIS 53 HIS 55 GLN 59 HIS 96 Mn HIS 53 HIS 55 GLN 59 HIS 94 HIS 96 HIS 53 HIS 55 GLN 59 HIS 96 HIS 55 GLN 59 HIS 96 HIS 53

3.2 3.6 4.3 3.9 3.3 2.1 2.3 2.1 2.5 2.2 3.3 3.7 3.6 3.5 2.9 3.1 2.9 3.4

3.3 3.5 3.4 3.6 2.7 2.4 2.4 2.0 2.5 2.5 3.3 3.6 3.7 3.7 3.3 3.0 3.3 3.5

Table 2. Angles of Opposing Corner Points of the Octahedron and Mn HIS 53/Mn/GLN 59 HIS 55/Mn/HIS 96 HIS 94/Mn/H2O

GtHNL (°)

GtHNL-A40H/V42T/Q110H (°)

166.9 163.6 158.5

167.1 171.4 165.4

Mn(II) sites in enzymes4−7 and also in certain Mn(II) model compounds (e.g., in refs 8−10). This spectrum is quite distinct from the well-known and ubiquitously occurring six-line pattern observed for S = 5/2 octahedral Mn(II) ion in frozen buffer (cf Figure S1) or in association with a wide variety of biological preparations. Mn(II) EPR is usually interpreted in terms of the spin Hamiltonian

Figure 1. (A) Stereoview of the metal binding site of GtHNL (PDB: 4BIF1a); (B) Twisted octahedron surrounding Mn of GtHNL; (C) Mn of GtHNL is below the square planar ligands and the water-Mn-His94 axis is bent by more than 20 degrees; (D) Twisted octahedron surrounding Mn of GtHNL-A40H/V42T/Q110H (PDB: 4UXA1b); (E) Mn of GtHNL-A40H/V42T/Q110H is below the square planar ligands and the water-Mn-His94 axis is bent by 15 degrees. The pictures were generated using the PyMOL Molecular Graphics System.

H = D[Sz 2 − S(S + 1)/3] + E(Sx 2 − Sy 2) + βB ·g ·S + S·A·I (1)

in which D is the axial and E is the rhombic zero-field splitting parameter, g is the g-tensor, and A is the metal hyperfine tensor, although several authors have reported on the necessity for quantitative analysis to include fourth-order zero-field terms,11 a quadrupole interaction term for the 55 Mn nucleus,12 rotation of diagonalizing axes systems for the different tensors,13 and/or an ad-hoc statistical distribution in zero-field parameters related to molecular conformational distribution, which can take rather complex forms indeed.14 The ensuing theoretically allowed large number of parameters combined with the fact that their magnitude cannot be unequivocally related to structure14 sentences interpretational efforts of the EPR spectra of these systems to remain somewhat of a “sporting technique” for the time being. We can, however, make several relevant qualitative observations. That the oxidation state of Mn in GtHNL is in fact 2+ is borne out by the observation that incubation with the general, lowpotential reductant dithionite has no effect on the spectrum (not shown). The spectrum has a distinct feature close to zero field, which becomes even more pronounced in the presence of any one of the three substrates HCN, benzaldehyde, or mandeloni-

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RESULTS AND DISCUSSION To probe the nature of manganese in GtHNL EPR (electron paramagnetic resonance) studies were performed. GtHNL was heterologously expressed in Escherichia coli. In the absence of manganese inactive apo-GtHNL was isolated. As expected apoGtHNL gave no EPR signal. Subsequent addition of Mn(II) yielded ∼100% loading with a metal to apoenzyme ratio of 1. When GtHNL was expressed in E. coli in the presence of Mn(II) a loading of 60% manganese in GtHNL was achieved.1 Both apoGtHNL loaded with manganese and GtHNL produced in the presence of manganese gave a multiline EPR spectrum extending over (and beyond) the full field range of the employed magnet (Figure 2), indicative for a high-spin system with zero-field interaction comparable in magnitude to that of the electronic Zeeman interaction. Spectra of rather similar shape have previously been observed for a small number of mononuclear 5082

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in the spin manifold approximately equal in energy to that of the microwave quantum. Under the simplifying model of the above spin Hamiltonian this interdoublet transition would imply |2D| ≅ hν for axial symmetry (E = 0) changing to |(4√7/3)D| ≅ hν for full rhombic symmetry (E = D/3), i.e. approximately 0.15 ≥ D ≥ 0.09 cm−1 for ν ≈ 9.4 GHz broadly consistent with six-coordinate N/O ligation.14 Quenching of orbital angular momentum in the half-filled dshell of S = 5/2 systems induces only minor g anisotropy (i.e., g ≈ ge = 2.002), and in the weak-field limit (hν ≪ |D|) this leads to the well-known effective g⊥ ≈ 6 (i.e., 3ge) feature from the mS = ±1/2 doublet found in, e.g., high-spin hemoproteins. When the ratio hν/|D| increases, the g = 6 feature moves to lower effective g values.16 Diagonalization of the energy matrix17 from the axial part of the Hamiltonian in eq 1 (ignoring hyperfine interaction) affords an effective g⊥ = 4.45 for D = 0.15 and ν ≈ 9.4 GHz, and at the corresponding field of ca. 1500 gauss we indeed detect a major feature in the Mn(II) spectra of GtHNL in the absence of substrate(s). In the presence of a rhombic term (E ≠ 0) this feature would be split into a gx absorption and a gy derivative feature; however, we only find the line to be asymmetric, which, again using energy matrix diagonalization, limits E ≤ 0.003 cm−1 (note, however, that the usually ignored zero-field term quartic in S18 of comparable strength would cause a similar splitting). Some asymmetry is present though, as judged from the complex pattern of 55Mn hyperfine lines due to interference of the 6-line patterns at gx and gy. The strength of the hyperfine interaction is more readily estimated from the hyperfine manifold at lower field (centered at ca. 750 gauss), a pattern that was also seen previously in an Mn(II) extradiol dioxygenase.7 The average splitting between the six hyperfine peaks is 92.4 gauss, i.e. a common value for Mn(II) complexes, and in particular close to the values reported for similar wide-field spectra from Mn(II) proteins and model compounds.5,7,10 When measured in regular X-band EPR, mononuclear Mn(II) sites (S = 5/2; I = 5/2) in proteins come in two very different classes: those with 2D ≪ hν and those with 2D ∼ hν, where D is the axial zero-field splitting parameter in the spin Hamiltonian, and hν is the energy of the microwave (ca. 0.3 cm−1 in X-band).4 The more common systems with 2D ≪ hν afford spectra that are dominated by a quasi-isotropic line close to g = 2.00 split into six 55 Mn nuclear hyperfine lines. Such a type of spectrum is also found for “free” Mn(II) in frozen buffer (cf. Figure S1a). Examples of the other class are less common in the literature because 2D ∼ hν implies very wide spectra typically extending over the full field range of the spectrometer with associated decreased amplitudes sometimes by 2 orders of magnitude compared to the 2D ≪ hν case.4−7 This increased zero-field interaction has been associated with significant distortion of Mn octahedral symmetry both in a MnN6 model compound9 and a mononuclear Mn(N/O)6 catechol dioxygenase5 each showing an EPR spectrum similar to those of GtHNL in Figure 2. A rather modest rhombicity in the spectra of GtHNL was estimated, above, from the asymmetric feature at g⊥ ≈ 4.45 to be limited to E < 0.003 cm−1; however, a symmetry-allowed (namely: cubic) quartic term in the zero-field interaction can not only give rise to a similar pattern (cf. ref 18) but with the proper sign can also largely anneal the effect of the term in E, which would imply a possible underestimation of the magnitude of E. Quantitative disentanglement of these subterms in the zero-field interaction has to our knowledge yet to be done for any biological system. To establish whether the unusual EPR spectrum is an essential feature of the catalytically active enzyme, inactive GtHNL-H96A

Figure 2. EPR spectra of wild type GtHNL. The enzyme is ca. 5 mg/mL, i.e. 0.33 mM monomer, in 20 mM Tris-HCl buffer, pH 7.5, and 200 mM NaCl. Trace a is apoenzyme reconstituted with Mn (Mn:enzyme = 1.0) and excess Mn removed (4.8 mg/mL); trace b is holo-enzyme (Mn:enzyme = 0.6) from cells grown with Mn; trace c is the enzyme of b with 8.5 mM HCN; trace d is the enzyme of b with 1 vol % benzaldehyde to a final concentration of 78 mM; trace e is the enzyme of b with 20 mM mandelonitrile. All spectra are scans of 327 s averaged over a 1−1.5 h period. The lower panel is an 18-scan average of the lowfield part of spectrum a to emphasize 6-line manganese nuclear hyperfine patterns. EPR conditions: microwave frequency, 9.39 GHz (9.61 GHz for the lower panel); microwave power, 80 mW; modulation frequency, 100 kHz; modulation amplitude, 8 gauss; temperature, 13 K.

trile (Figure 2) When the microwave, which is normally perpendicular to the magnetic field vector, is changed to parallel to the field, the spectral feature persists, while the rest of the spectrum is reduced (Figure S2), and this is characteristic for a semiforbidden transition15 consistent with a zero-field splitting 5083

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Figure 3. Stereoview of the overlay of the active site of GtHNL (green) and the variant GtHNL-A40H/V42T/Q110H (purple) (PDB: 4BIF and 4UXA). Mandelonitrile (pink) was docked in the active site of the variant as described elsewhere.1b The picture was generated using the PyMOL Molecular Graphics System.

was studied, in which the manganese would now be tetracoordinated. An EPR spectrum very similar to Mn(II) in frozen buffer was measured, the four remaining coordinating amino acids creating a very symmetric octahedral Mn(II) complex (Figure S1). As Mn(II) is typically not Lewis acidic enough to catalyze cyanohydrin syntheses3 this raised the question whether it is involved in the coordination of the substrates and product. Addition of any of the substrates HCN, benzaldehyde, or mandelonitrile induces a similar overall spectral change (Figure 2 c-e), whereas the addition of analogous compounds (benzoic acid, acetophenone, 2,2,2-trifluoroacetophenone, citronellal) have no effect (not shown), suggesting coordination of the substrates to manganese rather than non-specific binding to the protein. In that case each substrate would be expected to bind differently inducing nonuniform changes. The spectral change encompasses the following: (i) an increase in the intensity of the near-zero-field line, presumably due to the zero-field splitting becoming even closer to the value of hν; (ii) a considerable broadening (and perhaps even a split in the case of HCN) in the g ≈ 4.45 line accompanied by a general loss of resolution in the 55 Mn hyperfine patterns. Qualitatively, we interpret this observation in structural terms as an increase in flexibility (and possibly in rhombicity) of the coordination sphere of the Mn ion. Supporting this even stronger, some metal leaching occurred, as visible in the characteristic Mn(II) in frozen buffer EPR signals as seen in Figure 2 traces c and e, and in particular when mandelonitrile is added at once. This might be due to the bidentate character of mandelonitrile. When following the cyanohydrin formation or decomposition over time this did not significantly alter the EPR spectra providing solid evidence that the unusual octahedron of the active enzyme is a unique feature present throughout the catalytic cycle. No Mn(II) in the frozen buffer complex was formed during mandelonitrile synthesis, indicating its formation during mandelonitrile addition is an artifact due to local high mandelonitrile concentrations during its addition. When adding benzaldehyde to inactive GtHNL-H96A the EPR spectrum remained unchanged (Figure S1c), supporting that the coordination of the substrates to the Mn(II) in the enzyme is an essential step in this Lewis acid catalysis. Having established the oxidation state of manganese in active GtHNL as Mn(II) and the absence of redox activity during the

catalytic cycle this raises the question of the stability and character of the complex. Even careful addition of mandelonitrile could not completely suppress metal leaching (see above). Recently, GtHNL-A40H/V42T/Q110H was described with an improved specific activity. None of the altered amino acids acts as ligands of Mn(II); the metal is again octahedrally coordinated with water as the sixth ligand (Figures 1 and 3).1b Probing the stability of the metal binding with tridentate pyridine-2,6dicarboxylic acid (PDCA) reveals that manganese is bound more tightly than other metals. When the stability of the manganese complex of GtHNL is compared with that of GtHNL-A40H/ V42T/Q110H significantly less manganese is removed from the triple variant. This is also reflected in the activity retained (Figure 4). Clearly, GtHNL-A40H/V42T/Q110H is not only a better catalyst but the octahedral Mn(II) is bound more tightly.

Figure 4. Relative metal loading (luminol/PAR assay) and HNL activity of the different Apo preparations of GtHNL-A40H/V42T/Q110H compared to the original GtHNL-A40H/V42T/Q110H sample. Apo1: standard conditions = 10 mM PDCA, 26 h dialysis, Apo2:10 mM PDCA, 50 h dialysis, Apo3:20 mM PDCA, 26 h dialysis, Apo4:20 mM PDCA, 50 h dialysis. All manganese was removed from GtHNL under standard conditions.

EPR studies of this variant revealed essentially the same spectrum as with the wild type enzyme (Figure S3). When adding benzaldehyde or HCN virtually no change was visible, indicating an even higher rigidity of the Mn(II) complex in the conformation induced by the enzyme variant than in GtHNL. Upon addition of mandelonitrile essentially the same EPR spectrum was observed as before again indicating conformational rigidity. Moreover, no Mn(II) in frozen buffer complex was 5084

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formed when adding mandelonitrile, in line with the higher stability found for the Mn(II) binding (Figure 4). Overall this indicates that GtHNL and GtHNL-A40H/V42T/ Q110H are 17 electron octahedral Mn(II) complexes which act as Lewis acid in the cyanohydrin synthesis. Catalytic activity might be induced due to a twisted octahedral conformation. Support for this structural feature can be found in the twisted octahedral structure of Mn(II) complexes with a similar EPR spectrum (see EPR discussion above). When examining the overall catalysis the substrate aldehyde is proposed to coordinate via its oxygen atom to the Mn(II). This activates the carbonyl group for attack of the cyanide along the Bürgi-Dunitz angle. Deprotonation of the hydrogen cyanide by a suitably located base such as H106 will then initiate the cyanide addition. The relevance of this base for catalytic activity of GtHNL has earlier been demonstrated. When exchanging it for alanine, loss of activity was observed.1 Proton donation to the evolving oxyanion from H106 most likely via H96 yields the cyanohydrin that then leaves the active site (Scheme 1). In

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been supported by the Federal Ministry of Science, Research and Economy (BMWFW), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, the Government of Lower Austria, and ZIT - Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. We are grateful to Karin Reicher, MSc for protein purification.

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Scheme 1. Proposed Mechanism of Action of GtHNLa

a

Coordination of the carbonyl group to Mn(II) and subsequent attack of the cyanide ion along the Bürgi-Dunitz angle yields the chiral cyanohydrin. His106 and His96 are involved in deprotonation of HCN and in the protonation of the developing oxyanion.

GtHNL-A40H/V42T/Q110H additional histidines might improve the deprotonation step of the hydrogen cyanide and also, due to an extended hydrogen bond network, the shifting of the proton to the final product. In the reverse decomposition reaction a similar proton transfer can be envisaged.

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CONCLUSION In conclusion GtHNL is a Mn(II) containing enzyme. The metal acts as Lewis acid coordinating the substrates, thereby activating them. Tight binding of the metal is essential for its catalytic activity in the enantioselective synthesis of cyanohydrins.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscatal.6b01204. Experimental details on enzyme production, EPR measurements, and sample production (PDF)

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REFERENCES

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DOI: 10.1021/acscatal.6b01204 ACS Catal. 2016, 6, 5081−5085