PROTEINS: Structure, Function, and Genetics 19:343-347 (1994)

Crystallization and Preliminary X-Ray Diffraction Studies of Mandelonitrile From Almonds

Hanspeter Lauble,' Kirsten Muller,' Hermann Schindelin? Siegfried Forster,' and Franz Effenberger' 'Institut fur Organische Chemie und Isotopenforschung, Uniuersitat Stuttgart, 0-70569 Stuttgart, Germany and 'Institut fur Kristallographie, Freie Universitat Berlin, 0-14195 Berlin, Germany

ABSTRACT Single crystals of three differ- alyzing the dissociation of mandelonitrile into ben- ent isoenzymes of (R)-( +) mandelonitrile lyase zaldehyde and HCN.1° The protein is a single poly- (hydroxynitrile lyase) from almonds (Prunus peptide (M, 75,000) containing a FAD and is amygdalus) have been obtained by hanging heavily N-glycosylated with oligomannose units (H. drop vapor diffusion using polyethylene glycol Wajand, unpublished results). The amino-acid se- 4000 and isopropanol as co-precipitants. The quence of 12 tryptic peptides derived from MNL-A crystals belong to the monoclinic space group comprises 177 residues of the entire sequence (H. P2, with unit cell parameters a = 69.9, b = 95.1, Wajand, unpublished report). Recently the primary c = 95.6 A, and p = 118.5'. A complete set of structure of the black cherry (Prunus serotina) MNL diffraction data has been collected to 2.6 A res- has been deduced from its cDNA. l2 The sequence of olution on native crystals of isoenzyme 111. the tryptic peptides of MNL-A has been aligned with 0 1994 Wiley-Liss, Inc. respect to the black cherry sequence. It shows 98% identity and predicts a similar pat- Key words: hydroxynitrile lyase, flavoenzyme, tern. X-ray crystallography Key features of MNL-A function are: apo-MNL-A is catalytically activated with FAD'; con- INTRODUCTION taining reduced FAD is inactive, suggesting that the Hydroxynitrile (HNL) catalyze the ste- oxidation state of FAD is important with respect to reospecific retro-addition of a great number of ali- the in vivo modification of enzyme activity"; spec- phatic, aromatic, and heterocyclic cyanohydrines troscopic studies give evidence that the cofactor into hydrogencyanide and or ketones, re- FAD is bound near the catalytic site"; chemical spectively (Scheme I).',' The enzymatic reaction modification experiments implicate an essential cys- from left to right in Scheme I is a component of the tein-SH gr0~p.l~Although recent studies have es- cyanogenesis in plank3Release of hydrogencyanide tablished aspects of the mechanism of mandeloni- upon damage of cells in germinating or in trile lyase, the biological function of the FAD leaves is thought to be part of a protection strategy cofactor in the catalytic mechanism is still subject of against infectious pathogens. controverse discussion. l4 Two types of lyases that vary in subunit structure, In addition to its physiological role, MNL-A is im- cofactor specificity, and stereoselectivity of reaction portant as a biocatalyst in organic chernistry.l5J6 have been reported. HNL of Type I (families Ola- The enantioselective addition of HCN to aldehydes ~aceae,~Gramineae,5.6 and Luminaceae7) usually or ketones, respectively (e.g., reaction from right to exist in homodimeric or heterodimeric states with left in Scheme I), forms chiral cyanohydrines that subunit molecular weights between 22,000 and can be transformed into optically active a-hydroxy- 42,000 Da, respectively, of which up to 9% is carbo- carbonic acids or a-hydroxy-ketones, respectively, hydrate. These are cofactor independent. which are important intermediates for pharmaceu- The second type of HNL is found in different spe- ticals. cies of Rosaceae.' HNLs of this group are typically single chain glycoproteins with a molecular mass ranging from 50,000 to 80,000 Da, of which up to Abbrevations: PEG 4000, Polyethylene glycol 4000; HNL, 30% is carbohydrate. Enzymes exist in different iso- Hydroxynitrile lyases; FAD, Flavine-Adeninedinucleotide; forms and catalyze exclusively the decomposition of MNL-A, (R)-( + Mandelonitrile Lyase from Almonds; MNL- AI, Isoenzyme I of MNL-A; MNL-A 11, Isoenzyme I1 of MNL-A, cyanohydrines in the R-configuration. Most signifi- MNL-A 111, Isoenzyme I11 of MNL-A; MNL-A IV, Isoenzyme IV cant for Type I1 HNL is the presence of the cofactor of MNL-A. flavine-adeninedinucleotide(FAD), which is critical to the catalytic a~tivity.~ The best studied example of this conserved class of Received April 1, 1994; accepted April 6, 1994. Address reprint requests to Hanspeter Lauble, Institut fur enzymes is (R)-( +) mandelonitrile lyase (MNL-A, Organische Chemie und Isotopenforschung, Universitat Stutt- EC.4.1.2.10) from almonds (Prunus amygdalus) cat- gart, D-70569 Stuttgart, Germany.

0 1994 WILEY-LISS, INC. 344 H. LAUBLE ET AL. OH I -HCN //O \ R-C-CN \ R-C I +HCN \ H H

c yanohydrine

Scheme 1

To understand how MNL-A and other members of mM piperazine.HC1, pH 4.0, 10% polybuffer 74-HC1 this family of hydroxynitrile lyases interact stereo- (Pharmacia). Proteins were eluted with the same selectively with substrates, knowledge of their buffer; fractions with enzyme activity were dialyzed three-dimensional structure is crucial. In this paper against 20 mM piperazine.HC1, pH 5.5 and concen- we report crystals of almond mandelonitrile lyase trated. suitable for a high resolution crystal structure anal- Proteins were loaded onto a Q-Sepharose HP col- ysis. umn (Pharmacia; 3.5 x 10 cm) equilibrated with buffer C. The mandelonitrile lyase isoenzymes were MATERIALS AND METHODS eluted from the column as single peaks by applying Purification of Mandelonitrile Lyase a 2.9 1 linear NaCl gradient (0-250 mM NaC1) in Mandelonitrile lyase isoenzymes were purified to buffer C (Fig. 1A). Eluted fractions with enzymatic apparent homogeneity using various precipitation activity appeared homogeneous on Coomassie-blue and chromatographic steps. Protein concentrations stained sodium dodecyl sulfate/polyacrylamide gel were determind with the BCA Protein Assay electrophoresis (SDS/PAGE) (Fig. 1B). (Pierce). Mandelonitrile lyase activity was assayed The majority of the enzymatic activities were according to Bove and Conn.17 eluted at 68 mM NaCl [isoenzyme I11 (MNL-A 11111 Almond (Prunus amygdalus) seeds (2 kg) were and 80 mM NaCl [isoenzyme IV (MNL-A IV)], re- ground and defatted by repeated treatment with spectively. Minor portions of the activities were about 101 n-hexane. The powder was lyophilized, eluted at 55 mM NaCl [isoenzyme I (MNL-A I)] and resuspended in 41 water, and stirred for 24 h. The 62 mM NaCl [isoenzyme I1 (MNL-A 1113, respec- pH of the suspension was adjusted continuously to tively. Differences of the isoenzymes concerning 7.5 with 1M ammonia. The extract was centifuged at their elution properties might arise from variations 6,OOOg for 30 min. The supernatant was equili- in their primary structure and/or from heterogene- brated with 1M H,PO, to pH 5.4 and left overnight ity in their carbohydrate contents. The isoenzyme at 4°C. The resulting precipitate was again centri- samples were dialyzed against 10 mM Na-citrate, fuged at 6,OOOg for 30 min and the supernatant was pH 5.4, and concentrated to 20 mgiml using an Am- concentrated. icon concentrator with YMlO membrane. The enzyme extract was loaded onto a methyl p-hydroxybenzoat modified Eupergit C column" Crystallization Procedure and X-Ray (Rohm; 11.3 x 18 cm) equilibrated with 10 mM Na- Diffraction Studies citrate, pH 5.4, 25 mM NaCl (buffer A). After wash- The crystallization experiments were performed ing, the proteins were eluted with buffer A contain- at 20°C using the hanging drop vapor diffusion ing 500 mM NaC1. The peak fractions were pooled, method.lg Drops containing 4 p1 of 16 mg/ml protein dialyzed against 20 mM bis-Tris.HC1, pH 6.5 (buffer solution in 10 mM Na-citrate, pH 5.4, and 4 p1 of B), and loaded onto a Q-Sepharose HP column precipitant buffer were prepared on siliconized cov- (Pharmacia; 3.5 x 10 cm) equilibrated with buffer erslips and suspended over 0.7 ml precipitant buffer. B. After washing, a 11 linear gradient from 0 to 1 M The best reservoir conditions for crystallization NaCl in buffer B was applied. were 100 mM Tris.HC1, pH 7.4,20% PEG 4000,18% The peak fractions were combined, dialyzed isopropanol, 0.02% NaN,. against 20 mM piperazine.HC1, pH 5.5 (buffer C), For X-ray studies crystals were transformed into a and loaded onto a PBE-94 column (Pharmacia; 1 x stabilizing solution containing 100 mM Tris.HC1, 35 cm) equilibrated with buffer C. The pH gradient pH 7.4, 20% polyethylene glycol (PEG) 4,000, 23% for chromatofocusing was formed with 150 ml of 25 isopropanol, and carefully washed. Crystals were MANDELONITRILE LYASE X-RAY DIFFRACTION 345

A

150 175 200 225 250 rnh Retention Time

Fig. 1. Purification of mandelonitrile lyase (MNL-A) isoen- by the dashed line. B: SDS/l5% PAGE analysis of MNL-A isoen- zymes. A: Elution profile of MNL-A isoenzymes on Q-Sepharose zyrnes. The eluted, active peak fractions from the Q-Sepharose HP column. Active fractions from chromatofocusing PBE-94 col- were pooled and concentrated. lsoenzyme numbers are indicated umn were dialyzed against buffer C to remove polybuffer 74-HCI above the gel. Molecular masses of marker proteins are shown at and applied to Q-Sepharose HP column. The eluation profile is right. shown by the continuous line, and the NaCl gradient is indicated mounted into glass capillaries and the capillaries The unit cell dimensions of the crystals were de- were sealed with epoxy resin. termined from 155 reflections using MADNES.20 Crystal characterization was performed with an The data indicated that the crystals belong to the Enraf-Nonius FAST area detector system and monoclinic space groups of either P2 or P2, with MADNES2' using graphite monochromatized CuK, unit cell dimensions a = 69.9 A, b = 95.1 A, c = radiation produced with a FR 571 rotating anode 95.6 A, and p = 118.5". Further characterization of generator operating at 45 kV and 65 mA. The three- the crystals by precession photographs only showed dimensional data set was collected with a MAR-Re- reflections of type OkO to be present if k = 2n, com- search image plate and IPMOSFLM2' also using patible with space group P2,. Assuming two mole- graphite monochromatized CuK, radiation pro- cules of mandelonitrile lyase with a molecular duced with a FR 571 rotating anode generator oper- weight of 75,000 Da in the asymmetric unit, the vol- ating at 45 kV and 80 mA. The crystal to detector ume per unit mass is 2.1 A3/Da, within the range distance was 130 mm and data were collected in os- observed for a standard set of proteins.22 The cillation ranges of 1.5" with 20,000 countdimage. content of the crystals is about 40%. Moderate an- isotropy of x-ray intensities was observed with a RESULTS maximum resolution of 2.5 A along the a*-axis. Crystals of almond mandelonitrile lyase were Crystals are fairly resistant to decay upon x-ray ir- grown by hanging drop vapor diffusion. Initial crys- radiation. tallization conditions were established by variation A native x-ray data set was collected to 2.6 A res- of pH, protein concentration, temperature and na- olution from a single crystal. Data were recorded ture of percipitants. Small, thin, plate-shaped, yel- with a MAR Research image plate detector (Fig. 2B) low crystals of MNL-A 11, MNL-A 111, and MNL-A and processed with IPMOSFLM.21 A total of 55,287 IV appeared after 4 weeks using a reservoir of 100 observations was merged to give 31,388 unique re- mM Tris.HC1, pH 7.4, 20% PEG 4,000, 18% isopro- flections. Between 20 and 2.6 A resolution, 92.9% of panol, 0.02% NaN, (Fig. 2A). Crystals grew to their the possible reflections have been recorded with an maximum size of 0.8 mm x 0.3 mm x 0.2 mm Rsym(I) of 0.099. Completeness in the highest reso- within 2 months. They show a pronounced tendency lution shell (2.7-2.6 A) is 79% with an average I/a (I) towards twinning and, furthermore, crystallization greater than 3. Heavy atom derivatives are cur- trials using the conditions described were not al- rently being screened. ways successful. Using a fine grid of conditions cen- tered around the one described above has shown that the most critical factor for crystallization seems ACKNOWLEDGMENTS to be the concentration of the co-precipitant isopro- We are greatful to Prof. W. Saenger for using data panol. The crystallization of HNL-A isoenzyme, collection facilities in Berlin and to Dr. U. Heine- however, remains problematic mainly because of its mann for support, advice and critical reading of the extreme glycosylation. manuscript. This work was supported in part by a 346 H. LAUBLE ET AL.

Fig. 2. Photographs of crystals and diffraction pattern of mandelonitrile lyase of almonds (MNL-A). A: Monoclinic crystals of MNL-A grown from PEG 4,000-isopropanol. B: X-ray diffraction diagram of mandeloni- trile lyase isoenzyme Ill recorded on a MAR image plate detector; wavelength, 1.5418 A; crystal to film distance, 130 mm; exposure time, 10 min; oscillation range, 1.5”. Maximum resolution range in this picture is 2.3 A. grant from the Bundesministerium fur Forschung 2. Seigler, D.S. Isolation and characterization of naturally occuring cyanogenic compounds. Phytochemistry 14:9-29, und Technologie under A03U and AlOU. 1975. 3. Poulton, J.E. Cyanogenic compounds in plants and their REFERENCES toxic effects. In: “Handbook of Natural Toxins. Vol. I, Plant 1. Gerstner, E., Pfeil, E. Zur Kenntnis des Flavoenzyms Hy- Toxins.” Tu, A.T., Keeler, R.F., eds. New York: Marcel droxinitril-Lyase (D-Oxynitrilase).Hoppe-Seylers-Z. Phys- Dekker, 1983:117-157. iol. Chem. 353:271-286, 1972. 4. Kuroki, G.W., Conn, E.E. Mandelonitrile lyase from Xi- MANDELONITRILE LYASE X-RAY DIFFRACTION 347

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