Acetone cyanohydrin lyase from Manihot esculenta (cassava) is serologically distinct from other hydroxynitrile lyases

Plant Science 108 (1995) l-11

ELSEVIER

Acetone cyanohydrin lyase from Manihot esculenta (cassava) is serologically distinct from other hydroxynitrile lyases Harald Wajant*a, Siegfried F6rsterb, Heiner Biittinger”, Franz Effenbergerb, Klaus Pfizenmaier” aInstitute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany bInstitute of Organische Chemie. University of Stuttgart, Pfaffenwaldring 55. 70569 Stuttgart, Germany Received 29 November 1994; revision received 7 February 1995; accepted 20 March 1995

Abstract A non-flavoprotein hydroxynitrile lyase clearly different from other hydroxynitrile lyases was isolated from Munihot esculenta (cassava) by combined application of anion exchange chromatography and gel filtration. The purified protein was resolved, upon SDS-PAGE, as a single band of 30 kDa, while the molecular mass of the native enzyme, determined by gel filtration on Superdex 200 was 124 kDa. Diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride inhibited the activity of acetone cyanohydrin lyase, indicating an enzymatically important serine residue. Using immunological techniques, we demonstrate that there is no serological relationship between acetone cyanohydrin lyase from cassava and various other hydroxynitrile lyases. This supports the idea that hydroxynitrile lyases have independently evolved from various ancestoral proteins. The hydroxynitrile lyase from cassava is capable of catalyzing the addition of HCN to several aliphatic carbonyls in organic media, demonstrating the potential usefulness of this enzyme in stereoselective synthesis of aliphatic cyanohydrins, which are important building blocks in organic synthesis. Keywordr: Cyanogenesis; Hydroxynitrile

lyase; Convergent evolution

Abbreviations: CPDW, serine carboxypeptidase from wheat; ee, enantiomeric excess; LuHNL, hydroxynitrile lyase from Linum usitatissbnwn (acetone cyanohydrin lyase); MeHNL, hydroxynitrile lyaae from Manihot esculenta (acetone cyanohydrin lyase); PaHNL, hydroxynitrile lyase from Prunus amygdalum ((R)-mandelonitrile lyase); PhaHNL, hydroxynitrile lyaae from Phlebodeum aureum; SbHNL, hydroxynitrile lyase from Sorghum bicolor ((Q-phydroxy-mandelonitrile lyaae); SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis. Enzymes: CPDW (EC 3.4.16.1); LuHNL (EC 4.1.2.J); MeHNL (EC 4.1.2.37); PaHNL (EC 4.1.2.10); SbHNL (4.1.2.11). Corresponding author, Tel.: +49 711 6857446; Fax: +49 711 6857484.

1. Introduction Cyanogenesis, the release of HCN, has been described for more than 2500 species of higher plants [l]. Release of cyanide occurs from cyanogenic precursors - mostly cyanogenic glycosides, in which the unstable cyano group of an or-hydroxynitrile is stabilized by 0-&glycosidic linkage to sugars [2]. Cyanogenesis occurs after mechanical tissue disruption or attack by herbivores or fungi [3]. In most cases, the initial step of cyanogenesis is the /3-glucosidase catalyzed

0168~9452/95k$O9.500 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-9452(95)04118-E

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H. Wajant et al. /Plant

hydrolysis of cyanogenic glycosides to cyanohydrin and glucose. Subsequently, the unstable arhydroxynitrile decomposes spontaneously or by the action of a stereospecific hydroxynitrile lyase to cyanide and aldehyde or ketone. However, in some plants, the catabolism of cyanogenic glycosides by sequential action of /3-glucosidase and hydroxynitrile lyase does not only occur during cyanogenesis but also during germination without detectable release of HCN [4,5]. Based on their FAD-content, HNLs are divided into flavo- and non-flavoprotein HNLs [6]. Flavoprotein HNLs are only described for members of the Rosaceae family [7], whereas nonflavoprotein HNLs are found in several families of higher plants [6]. All flavoprotein HNLs have (R)mandelonitrile as in vivo substrate, are of similar molecular weight and are serologically related [8- 111. Recently, cDNA cloning of mandelonitrile lyase from Prunus serotina showed that fIavoprotein HNLs contain a region of extensive homology to several dehydrogenases (MDL, residues 180325), where the degree of similarity averaged 77% [12]. In contrast to the flavoprotein HNLs, nonflavoprotein HNLs form a rather heterogeneous group of enzymes. They differ in size, glycosylation pattern, subunit composition and substrate specificity [6]. The amino acid sequence of HNL from Sorghum bicolor (SbHNL), deduced from cDNA clones, has revealed that the tetrameric structure of SbHNL is the product of a posttranslational processing of a single gene product and shows significant sequence homologies to serine carboxypeptidases [ 131. Western blotting experiments also revealed cross reaction between carboxypeptidase from wheat and anti-SbHNL antisera (131. Serine carboxypeptidases are examples of both convergent and divergent evolution. On one hand, they contain a catalytical triad (Ser, Asp, His) found in three groups of phylogenetic independently evolved proteases [ 141. This catalytical triad is also conserved in SbHNL [ 131. On the other hand, serine carboxypeptidases share a conserved structural motif, the so-called o//3 hydrolase fold, with a series of non-proteolytic enzymes [ 15- 181.Taking into account that the structure of SbHNL differs from all other HNLs described so far, it seems conceivable that HNLs have evolved from various ancestoral enzymes.

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HNLs have been employed as catalysts for enantioselective addition of HCN to aldehydes or ketones, yielding a-hydroxynitriles in very high enantiomeric purity [ 19,201. Optically active IXhydroxynitriles are interesting intermediates for the synthesis of ar-hydroxy acids, ar-hydroxy ketones or /3-ethanolamines, all of which are important building blocks in organic synthesis [20,21]. In the present study, we describe the puritication of acetone cyanohydrin lyase from Manihot escufenla Crantz (cassava) and show that this enzyme possesses a serine residue important for enzymatic activity. Serological comparison to other HNLs demonstrates that acetone cyanohydrin lyase from Manihot esculenta (MeHNL) is distinct from other HNLs, and probably defines a novel HNL subgroup. We also report the potential use of MeHNL as chiral catalyst for the synthesis of optically active cr-hydroxynitriles. 2. Materials and methods 2. I. Chemicals Except where noted, chemicals were purchased from Sigma. Carboxypeptidase from wheat (CPDW) and protein standards for SDS-PAGE and gel filtration were also from Sigma (Deisenhofen, Germany). Anti-trypsin and alkaline phosphatase conjugated anti-mouse (IgG+IgM) antibody were obtained from Boehringer Mannheim (Germany). Adjuvans was obtained from Linaris GmbH (Bettingen, Germany). Chromatography resins were from Pharmacia LKB Biotechnology Inc. (Freiburg, Germany) and the bicinchoninic acid (BCA) protein assay kit was from Pierce Chemical Co. (Rockford, IL, USA). 2.2. Enzyme assays The activity of acetone cyanohydrin lyase from cassava (MeHNL) and HNL from Linum usitatissimum (LuHNL) were assayed by measuring the HCN liberated during decomposition of acetone cyanohydrin according to Selmar et al. [22]. The activities of HNLs from Sorghum bicolor (SbHNL), Prunus amygaizhun (PaHNL) and Phlebodium aureum (PhaHNL) were determined, using p-hydroxymandelonitrile or mandelonitrile as substrate, according to Bove and Conn [23].

H. Wajant et al. /Plant Science 108 (1995) I-11

2.3. Protein assay

Protein concentrations were measured by a modified Lowry assay (BCA assay kit, Pierce Chemical Co.) with bovine serum albumin as standard. 2.4. Purification of acetone cyanohydrin lyase from cassava (MeHNL) The purification procedure, except the FPLC steps, was carried out on ice or at 4°C. Step I: Protein extraction. Dehydrated leaves of Manihot esculenta were frozen in liquid N, and were homogenized by grinding in a mortar. The resulting powder was extracted on ice with 25 mM sodium acetate buffer containing 0.5 M NaCI for 1 h. Subsequently, the solid material was collected by centrifugation in a Beckmann ultra centrifuge (4°C 30 min, 40 000 x g, rotor Ti45) and reextracted as described. Step 2: Partial removal of secondary plant products and extremely acidic proteins. The combined supernatants from the protein extraction were mixed with l/20 vol. of Q-sepharose FF equilibrated in 25 mM sodium acetate, pH 5.8, stirred gently for 15 min and filtrated through tilter paper. Subsequently, the filtrate was dialyzed for 12-18 h with 2 changes against 25 mM sodium acetate, pH 5.8 and was finally prepared for FPLC applications by filtration through a 0.45 pm membrane (Schleicher & Schuell). Step 3: Anion exchange chromatography on Mono Q at pH 5.8. The protein sample was loaded onto a Mono Q HR 5/5 column, equilibrated in 20 mM sodium acetate, pH 5.8. Thereafter, the column was rinsed with the above buffer until the extinction reached the base line of column equilibration. Subsequently, bound protein was eluted in a 30 ml linear gradient of O-O.5 M NaCl in 20 mM sodium acetate, pH 5.8, at a flow rate of 1 ml/min. This and all other FPLC operations were performed at room temperature with ice-cold buffers. Fractions were collected in an ice-cooled rack. The fractions were assayed for acetone cyanohydrin lyase activity and samples were pooled based on their specific activity. Step 4: Gel filtration chromatography. 5-10 ml aliquots of the protein sample were applied to a HiLoad 26/60 Superdex 200 prep grade column (Pharmacia), equilibrated in 20 mM Tris, pH 7.5,

3

200 mM NaCl. Proteins were eluted in 20 mM Tris, pH 7.5, 200 mM NaCl at a flow rate of 1 ml/mm and fractions with high specific activity were pooled. Step 5: Anion exchange chromatography on Mono Q at pH 7.5. Protein samples from gel tiltra-

tion chromatography were diluted 1:3 with Hz0 and applied to a MonoQ HR 5/5 column, equilibrated in 20 mM Tris, pH 7.5. After sample application, the column was washed with 10 ml of starting buffer. Elution of bound proteins was carried out with a 30 ml linear gradient of O-500 mM NaCl in 20 mM Tris, pH 7.5, at a flow rate of 1 ml/min. MeHNL activity eluted as one peak at 140- 160 mM NaCl. 2.5. SDS-PAGE Electrophoresis was performed according to Laemmli et al. [24]. Acrylamide gels were silver stained using the procedure described by Blum et al. [25]. 2.6. Generation of polyclonal antibodies Preimrnune sera were collected from Balb/c mice before immunization with either MeHNL, wheat carboxypeptidase or PaHNL. Immunization was carried out with IO-30 pg of each purified enzyme emulsified with ABM-S adjuvant. Booster injections were started 3 weeks later with the same amount of antigen, emulsified in AMB-N. Booster injections were repeated 4-6 times in 2-week intervals. Antisera were diluted 1: 10 in phosphate buffered saline and stored in 500 ~1 aliquots at -20°C to avoid multiple cycles of freezing and thawing. 2.7. Immunoblotting Proteins were resolved by SDS-PAGE and electroblotted onto a nitrocellulose membrane (Schleicher & Schuell) by the method of Towbin et al. [26]. The blotted membrane was blocked in Tris buffered saline (20 mM Tris, pH 7.5, 0.5 M NaCl) containing 3% gelatin. After one wash with Tris buffered saline (5 min), the nitrocellulose membrane was incubated with anti-HNL or antiCPDW antisera at a 1:lOOOdilution (1 h, 22”C, Tris buffered saline supplemented with 0.5% gelatin). Subsequently, the antigen-antibody complex was visualized using alkaline phosphatase conjugated anti-mouse (IgG+IgM) antibody (0.3

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Table 1 Purification of acetone cyanohydrin lyase from cassava Step

Total protein (mg)

Total activity (W

Specific activity Vmg)

Yield (%)

Purification (-fold)

Crude extract Q-sepharose treatment Mono Q pH 5.8 Gel filtration Mono Q pH 7.5

1240 120 30 6.2 1.2

475 464 233 167 110

0.38 0.64 1.11 26.9 91.6

100 98 49 35 23

1.68 20.4 70.8 241

&nl, 1 h, RT, Tris buffered saline, 0.5 % gelatine) with a substrate combination of 0.01% 5-bromo-4chloro-indolyl phosphate (BCIP), 0.02% nitro blue tetrazolium (NBT) and 4 mM MgClp.

a 1.0 3

1.0

0.7 i-

0.9 0.9 0.7

2.8. Immunoprecipitation HNL samples (150 ~1) were incubated with various amounts of antisera or preimmune sera (5-20 ~1) in 20 mM Tris, pH 7.5 with 200 mM NaCl at 22°C for l-6 h. Subsequently, the antigen-antibody complex was bound to protein G beads (50 ~1) and removed from the sample by centrifugation. Finally, the supematant was assayed for HNL activity. 2.9. Investigation

of substrate

0.6 0.5

z

0.4 3 0.3 = 0.2 0.1 0.0

\

I

0

5

’

I

I

,

10

15

20

’

0.0

elution volume [mr]

M A

range in organic

B

media

To immobilize acetone cyanohydrin lyase, 250 ~1 of enzyme (160 U/ml) was added to 5 ml of a suspension of avicel-cellulose (250 mg) previously stirred for 1 h in 20 mM acetate buffer, pH 3.4. Subsequently, the mixture was shaken at room temperature for 10 min, filtered and the immobilized enzyme resuspended in diisopropyl ether (4 ml). After addition of the appropriate aldehyde (0.5 mmol) and HCN (2.6 mmol), the mixture was shaken for 4.5 h. After removal of the immobilized enzyme, the filtrate was concentrated to yield the cyanohydrins. Finally, yield of reactions were determined by NMR and optical purity (in % ee) was measured by GC analysis as O-acetylderivatives. 3. Results 3. I. Purification of acetone cyanohydrin lyase from cassava (MeHNL) The results of a typical purification

are sum-

20.1

- m

14.4

- m

Fig. 1. (a) Anion exchange chromatography at pH 7.5. Fractions with high specific activity obtained after anion exchange chromatography at pH 5.8 and gel filtration were chromatographed on a Mono Q HR 515anion exchange chromatography column, equilibrated in 20 mM sodium acetate, pH 7.5. Elution was carried out with a linear 30 ml gradient of O-400 mM NaCl in starting buffer. Fractions of 0.5 ml were collected and assayed for acetone cyanohydrin lyase activity. (b) Analysis of MeHNL by SDS-PAGE. 100 ng aliquots of MeHNL were treated with DTT (40 mM)ISDS (A) or SDS only (B) and subjected to SDS-PAGE (13.5% polyacrylamide gel, silver stained). Molecular mass markers (M) used were phosphorylase B (97 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and lactalbumin (14.4 kDa).

H. Wajant et al. /Plant Science 108 (1995) l-11

marized in Table 1 and were reproduced with different batches of plant material. The final purification step, anion exchange chromatography on Mono Q HR 51’5at pH 7.5 is shown in Fig. la. The

5

symmetric peak of absorbance at 280 nm matched with the peak of activity. Analysis of peak fractions in reducing and non-reducing SDS-PAGE revealed, in silver stained gels, a single band of

1.0 0.8 0.6 0.4 0.2

‘0

I

0.0 180

200

220

I

I

I

I

I

240

wavelength [nm]

elution volume [ml]

100

100 80 -

d ?\

60 -

(

80 -

’

/

60 -

/I f

40 -

I

I

0

4

5

40 -

/

20 0

-3

*a’

PH

6

’

I

I

I

I

I

0

IO

20

30

40

50

Temperature [“Cl

Fig. 2. (a) Determination of molecular mass of native MeHNL by gel filtration on Superdex 200. 5 ml of purified MeHNL (SO@/ml) were applied to a HiLoad 26/60 Superdex 200 prep grade column, equilibrated in 20 mM Tris, pH 7.5, 200 mM NaCI, at a flow rate of I mVmin. Molecular mass standards (0) were (1) catalase 232 kDa, (2) aldolase 158 kDa, (3) bovine serum albumin 67 kDa, (4) ovalbumin 43 kDa, (5) carbonic anhydrase 30 kDa, (b) trypsin inhibitor 20.1 kDa. MeHNL eluted at a volume corresponding to 124 kDa (0, indicated by an arrow). (b) Absorption spectrum of purified MeHNL. The spectrum was determined with a protein concentration of 50 &ml in 10 mM sodium acetate, pH 5.2. (c) Effect of pH on acetone cyanohydrin lyase activity. Enzyme activity was determined in 50 mM acetate or citrate buffers (0) with IO mM acetone cyanohydrin. Non-enzymatic, base catalyzed breakdown of substrate was corrected. (d) Effect of temperature on acetone cyanohydrin lyase activity. Enzyme assays as described under Materials and methods were carried out with purified enzyme (5 U/ml) at various temperatures at pH 5.4 (0) for 10 min. The base catalyzed cleavage of acetone cyanohydrin was corrected.

H. Wajanr et al. /Plant

6

about 30 kDa (Fig. lb), indicating homogeneity of MeHNL. Purification of MeHNL resulted in an enzyme preparation of 91.6 U/mg with a 241-fold purification compared to the crude extract (Table 1). The specific activity of MeHNL is rather low if compared with the flavoprotein PaHNL, but is in the same range as described for other nonflavoprotein HNLs. The molecular mass of native MeHNL, determined by gel filtration on HiLoad 26160 superdex 200 prep grade column, was found to be 124 kDa (Fig. 2a), suggesting a homotetrameric structure of MeHNL. 3.2. Spectral properties of MeHNL Absorbance spectra of purified MeHNL was measured over wavelengths of 200-500 nm. An absorbance maximum at 278 nm was found, which is characteristic of aromatic amino acids in the enzyme. No further maxima, suggestive of prosthetic groups or other cofactors, were found. In particular, there was no FAD-typical absorbance maximum at 390 nm, indicating that MeHNL is a non-flavoprotein HNL (Fig. 2b).

Science 108 (1995) I-11

3.3. Effect of pH and temperature on iUeHNL activity

The influence of pH on MeHNL activity was determined between pH 2 and 6 (Fig. 2~). At higher pH, reliable determination of enzyme activity was not possible, due to the rapid base catalyzed non-enzymatic dissociation of acetone cyanohydrin. Optimal activity of acetone cyanohydrin lyase was obtained at pH 5.5, which is comparable to other HNLs [6].

Table 3 Acetone cyanohydtin lyase catalyzed synthesis of cyanohydrins from carbonyls and HCN Carbonyl Benzaldehyde

Reagent

Concentration (mM)

Inhibition (%)

DFP

2 0.5 2 0.5 5 2 2 5 5 5 I 1 1 1 I

85 70 40 28 0 0 0 0 0 0 0 0 0 0 0

PMSF Benzamidine TLCK TPCK DI-T EDTA EGTA CaCI, MgCl, MnCls ZnCl, cuso,

DFP, diisopropyl fluorophosphate; PMSF, phenylmethylsulfonyl fluoride; TLCK, N-ptosyl+lysine chloromethyl ketone; TPCK, L-I-tosyl-amido-2-phenylethyl chlorometyl ketone; DTf, dithiothreitol.

ee (%)

34

90

70

88

90

38

n.d.

70

n.d.

74

Ii

Butanal Table 2 Effects of various reagents on acetone cyanohydrin lyase activity

Yield (%)

Ii

4

0

Thiophen-3-aldehyde

isi

H

% 0

Thiophen-Zaldehyde H

Kv 0

Reactions were carried out with immobilized enzyme in diisopropyl ether for I h at room temperature. Yield was determined by NMR and ee was measured by GC analysis of Qacetyl-derivatives.

H. Wajant et al. /Plant

Acetone cyanohydrin lyase from cassava was active over a broad temperature range. Activity generally increased up to 4O’C and leveled off between 40-50°C (Fig. 2d).

a

Science 108 (1995)

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3.4. Effects of various additives on MeHNL activity The influence of various additives on acetone cyanohydrin is summarized in Table 2. Metal ions, reducing and chelating agents had no effect on ac-

I

Aa

I-11

II

Bb

b

ABCDEF

ABCDEF

ABCDEF

Fig. 3. (a) Characterization of antisera. Purified antigens (MeHNL (A), PaHNL (B); - 100 ng each) or corresponding crude extracts (13.5%).Proteins were subsequently electroblotted onto nitrocellulose and probed with anti-MeHNL antisera (I) or anti PaHNL antisera (II). The final dilution of antisera was 1:lOOO.Antigen-antibody complexes were visualized with alkaline phosphatase conjugated anti-mouse (IgG+IgM) antibody and BCIP and NBT as substrates. (b) Analysis of immunological relationship of HNLs. In order to determine the phylogenetic relationship of various HNLs (MeHNL (A), SbHNL (B), PaHNL (C), LuHNL (D), CPDW (E) and PhaHNL (F)), enzyme samples were resolved by SDS-PAGE, electroblotted onto nitrocellulose and treated with antisera against MeHNL (I), SbHNL (II) or PaHNL (III). Either 100 ng of purified enzyme or 0.2 U activity in crude extract were applied on the acrylamide gel (13.5%). Immunoblotting was carried out similar to Fig. 3b.

(Manihot esculenta (a), Prums amygaidus (b), 2- 10 pg of total protein) were subjected to SDS-PAGE

H. Wajant et al. /Plant Science IO8 (1995) I-11

8 Table 4 Immunoprecipitation of various HNLs Antisera

Anti-MeHNL Anti-SbHNL Anti-PaHNL

Remaining activity in supematant (%) Manihot esculenta

Sorghum bicolor

Prunus amygdalus

Linum usitatissimum

Phlebodium aureum

20 100 loo

100 10 100

loo 100 15

100 100 95

100 100 100

To reveal potential serological relationships, HNLs were treated with distinct anti-HNL antisera. Subsequently, antigen-antibody complexes were bound to protein G sepharose beads and removed by centrifugation. The remaining enzyme activities in supernatants were determined.

tivity, whereas the typical serine protease inhibitors diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride were strong inhibitors. This suggested the involvement of a catalytically important serine residue.

munological relationship between any of the HNLs, whereas the previously reported [ 131 crossreactivity of anti-SbHNL antisera and CPDW was clearly evident (Fig. 3b). 4. Discussion

3.5. Enzyme catalyzed synthesis of cyanohydrins in organic media We have investigated the substrate range of acetone cyanohydrin lyase from cassava (MeHNL), immobilized on avicel-cellulose, in diisopropyl ether. As summarized in Table 3, MeHNL accepts aliphatic, aromatic and heterocyclic carbonyls as substrates. Moreover, MeHNL possesses a strong stereoselectivity for (S)-cyanohydrins. 3.6. Immunological characterization In an initial search for relationships of MeHNL to other HNLs, we investigated immunological crossreactivity of MeHNL with various other HNLs (SbHNL, PaHNL, LuHNL, PhaHNL) and CPDW. The latter contains high sequence homologies to SbHNL (- 50% [ 131). The specificity of the employed antisera against MeHNL and PaHNL was controlled by SDS-PAGE and immunoblotting of purified antigens and the corresponding crude extracts (Fig. 3a). Specificity and properties of anti-SbHNL antisera have been described elsewhere [27]. For determination of potential crossreactivities, various concentrations of enzymes and antisera were tested by immunoblotting (Fig. 3b) and immunoprecipitation (Table 4). With these methods, we could not detect an im-

We report here the purification and characterization of hydroxynitrile lyase from Manihot esculenta, which acts on aliphatic substrates. Like hydroxynitrile lyases from Sorghum bicolor [23], Linum usitatissimum [28] and Ximenia americana [29], acetone cyanohydrin lyase from cassava is not a flavoprotein. Our data suggest that native MeHNL is a homotetrameric enzyme with an apparent molecular mass of approximately 124 kDa and subunits of 30 kDa. Accordingly, acetone cyanohydrin lyase from cassava is different in its composition from other non-flavoprotein HNLs, which are either heterotetrameric (SbHNL) [30], homodimeric (LuHNL) [28] or monomeric holoenzymes (XaHNL) [29]. Recently, we found an unexpectedly high amount of sequence homology between SbHNL and serine carboxypeptidases [13]. Due to the lack of common biochemical properties of non-flavoprotein HNLs, and faced with the similarities between SbHNL and serine carboxypeptidases, we discussed the possibility of independent evolution of HNLs from different ancestoral proteins [13]. To further evaluate this idea, we have investigated the serological relationship of MeHNL and various other HNLs. Independent immunoblotting and immunoprecipit-

Molecular weight of subunits Reaction with anti-MeHNL antisera Reaction with anti-SbHNL antisera Reaction with anti-PaHNL antisera References

structure

Family FAD Carbohydrate Substrate

-I-+++

El

-

[301

-

-

Rosaceae yes yes (R)-mandelonitrile monomer 6oooo

PaHNL

H-i+

yes (S)-p-hydroxymandelonitrile heterotetramer 33 ooo, 18000 -

Gramineae no

SbHNL

-

++++

Euphorbiaceae no no acetone cyanohydrin homotetramer 33 ooo

MeHNL

Table 5 Properties of various HNLs and serine carboxypeptidase from wheat

[281

Linaceae no no acetone cyanohydrin dimer 42OC0

LuHNL

-

Polypodiaceae no n.d. (R)-mandelonitrile nd. n.d.

PhaHNL

[291

-

-

-

:z-mandelonitrile monomer 33 ooo

Olacaceae no

XaHNL

[I41

+++

heterotetramer 33 ooo, 18000 -

yes Peptides

Gramineae no

CPDW

10

H. Wajant et al. /Plant Seienee 108 (1995) 1-I 1

ation analysis provided no evidence for a serological relationship between the HNLs from Manihot esculenta, Prunus amygdalum, Sorghum bicolor, Linum usitatissimum and Phlebodium aureum. In particular, there was no detectable rela-

tionship between acetone cyanohydrin lyase from cassava, which is an (5’)-HNL, and acetone cyanohydrin lyase from flax, which is an (R)-HNL [31]. These results suggest that HNLs of higher plants share, if any, only a limited number of conserved epitopes. In contrast, a structural relationship between SbHNL and serine carboxypeptidases exists [ 131. Moreover, serine carboxypeptidases share the presence of a catalytic triad not only with SbHNL, but also with two groups of serine proteases, namely the subtilisin and chymotrypsin family. The latter finding was taken as evidence for convergent evolution [14]. Furthermore, serine carboxypeptidases and SbHNL also share a conserved structural motif, the o//3 hydrolase fold, with various other enzymes [ 141. Hence, the question arose if other HNLs, structurally unrelated to SbHNL, contain one or all of these characteristic markers: (1) a catalytic triad, (2) homologies to subtilisin or chymotrypsin, (3) an arlfl hydrolase fold. The available data summarized in Table 5 suggest that acetone cyanohydrin lyase from cassava shares no structural properties with other HNLs. In particular, no serological relationship to other HNLs of higher plants was found. Using various well known serine protease inhibitors, we therefore investigated whether or not acetone cyanohydrin lyase from cassava also contains the catalytic triad Ser, Asp, His. MeHNL activity was almost completely inhibited by diisopropyl fluorophosphate and was significantly reduced by phenylmethanesulfonyl fluoride, two agents which act by modifying enzymatically important serine residues. These data are in accordance with the existence of a catalytic triad in MeHNL and support the idea that HNLs have independently evolved from different ancestoral proteins (convergent evolution). While this manuscript was in preparation, the cDNA sequence of MeHNL was published by Hughes et al. [32J. Their data show no obvious se-

quence homology to other proteins of known function. In particular, there were no homologies to SbHNL or PaHNL. This result is in good accordance with the idea of convergent evolution of HNLs. During the last few years several groups have demonstrated the potential use of HNLs in organic synthesis [19-21,311. In reversion of the natural direction of HNL action, these enzymes can be used to produce cyanohydrins, important building blocks in organic synthesis, by addition of HCN to various carbonyls [21]. As HNLs are active in organic media, where the base catalyzed, nonstereoselective addition of HCN to carbonyls is fully suppressed, the synthesis of cyanohydrins can be achieved at a very high optical purity [ 191. The use of organic media is of further advantage, as it bypasses problems of solubility of many interesting substrates. For synthesis of (S)-cyanohydrins, SbHNL is mostly used [20,21] while the synthesis of (R)-cyanohydrins is performed with (R)-mandelonitrile lyases from Rosaceae (mainly aromatic cyanohydrins) [ 19,211 and acetone cyanohydrin lyase from Linum (aliphatic cyanohydrins) [31]. We report here that the HNL from cassava is also active in organic media. We have successfully used acetone cyanohydrin lyase from cassava to synthesize aliphatic and aromatic (5’)cyanohydrins of high optical purity. Already under non-optimized conditions, an enatiomeric excess comparable to that published for LuHNL [31] and PaHNL [19] was achieved. These results demonstrate that acetone cyanohydrin lyase from cassava is a useful supplement to SbHNL for synthesis of aliphatic (S)-cyanohydrins, which are poorly accepted by SbHNL. Further studies on the substrate range of MeHNL will reveal its potential usefulness for biosynthesis of organic compounds. Acknowledgments This work was supported by the Bundesministerium fur Forschung und Technologie, Germany, grant No. A03U-ZSP Stuttgart. We wish to thank Dr. Dirk Selmar for providing us with plant material from Manihot esculenta and Phlebodium aureum .

H. Wajanl et al. /Plant Science 108 (1995) I-I I

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