Molecular cloning of the full-length cDNA of (S)-hydroxynitrile lyase from Hevea brasiliensis. Functional expression in Escherichia coli and Saccharomyces cerevisiae and identification of an active site residue.

The full-length cDNA of (S)-hydroxynitrile lyase (Hnl) from leaves of Hevea brasiliensis (tropical rubber tree) was cloned by an immunoscreening and sequenced. Hnl from H. brasiliensis is involved in the biodegradation of cyanogenic glycosides and also catalyzes the stereospecific synthesis of aliphatic, aromatic, and heterocyclic cyanohydrins, which are important as precursors for pharmaceutical compounds. The open reading frame identified in a 1.1-kilobase cDNA fragment codes for a protein of 257 amino acids with a predicted molecular mass of 29.2 kDa. The derived protein sequence is closely related to the (S)-hydroxynitrile lyase from Manihot esculenta (Cassava) and also shows significant homology to two proteins of Oryza sativa with as yet unknown enzymatic function. The H. brasiliensis protein was expressed in Escherichia coli and Saccharomyces cerevisiae and isolated in an active form from the respective soluble fractions. Replacement of cysteine 81 by serine drastically reduced activity of the heterologous enzyme, suggesting a role for this amino acid residue in the catalytic action of Hnl.

The full-length cDNA of (S)-hydroxynitrile lyase (Hnl) from leaves of Hevea brasiliensis (tropical rubber tree) was cloned by an immunoscreening and sequenced. Hnl from H. brasiliensis is involved in the biodegradation of cyanogenic glycosides and also catalyzes the stereospecific synthesis of aliphatic, aromatic, and heterocyclic cyanohydrins, which are important as precursors for pharmaceutical compounds. The open reading frame identified in a 1.1-kilobase cDNA fragment codes for a protein of 257 amino acids with a predicted molecular mass of 29.2 kDa. The derived protein sequence is closely related to the (S)-hydroxynitrile lyase from Manihot esculenta (Cassava) and also shows significant homology to two proteins of Oryza sativa with as yet unknown enzymatic function. The H. brasiliensis protein was expressed in Escherichia coli and Saccharomyces cerevisiae and isolated in an active form from the respective soluble fractions. Replacement of cysteine 81 by serine drastically reduced activity of the heterologous enzyme, suggesting a role for this amino acid residue in the catalytic action of Hnl.
Hydroxynitrile lyases (Hnls) 1 are involved in a process termed "cyanogenesis" and catalyze the final step in the biodegradation pathway of cyanogenic glycosides in plant cells. The degradation starts with the hydrolysis of cyanogenic glycosides by ␤-glycosidases (1) to form the aglycons, ␣-hydroxynitriles, or cyanohydrins. The unstable aglycons are then cleaved into HCN and the corresponding carbonyl component by Hnl. Hydroxynitrile lyases were first described by Rosenthaler (2) at the beginning of this century. HCN released by Hnl can either serve as a repellent factor to predators (3) or as a susceptibility component of plants to fungal attack (4) when produced at high local concentrations. A potential physiological role for cyanogenic glycosides as storage compounds for nitrogen has also been suggested (5). HCN released from cyanohydrins can be fixed by ␤-cyanoalanine synthase and then used for amino acid biosynthesis in plants. ␣-Hydroxynitrile lyases have attracted the interest of several laboratories, as they can be used as biocatalysts for the production of enantiomerically pure stereoisomers of cyanohydrins (6). Hnls catalyze the stereospecific retroaddition of a great number of aliphatic, aromatic, and heterocyclic cyanohydrins from HCN and aldehydes or ketones, respectively. Chiral cyanohydrins are the starting material for the synthesis of many important pharmacologically active compounds (6).
␣-Hydroxynitrile lyases have been isolated and partially characterized from a variety of species and assigned to two classes, depending on their properties. The first class of Hnls, isolated from Rosaceae, are single chain glycoproteins with up to 30% carbohydrate content and a molecular mass between 50 and 80 kDa. These enzymes are cofactor-dependent, with FAD as the prosthetic group, and accept (R)(ϩ)-mandelonitrile as their substrate. (R)-Hnls from several species of Rosaceae appear to be related to flavoproteins, such as alcohol or glucose dehydrogenases, or glucose oxidases (7). A cysteine residue has been shown to be required for catalytic activity (8,9), whereas the function of the FAD prosthetic group is still a matter of debate. These enzymes are present in relatively high concentrations in seed tissues and require only 5-10-fold purification to reach homogeneity.
The second class of Hnls are cofactor-independent with a subunit molecular mass of 28 -42 kDa. Active enzymes of this type have been isolated from different families (Olacaceae, Gramineae, Linaceae, Euphorbiaceae), contain up to 9% carbohydrate, and adopt homo-or heterodimeric conformations in vivo. Class II Hnls accept (S)-mandelonitrile, 4-OH-(S)-mandelonitrile, acetone cyanohydrin, or (R)-2-butanone cyanohydrin as substrates (see Table I) and require more than 100-fold purification to reach homogeneity. (S)-Hydroxynitrile lyase from Sorghum bicolor (Gramineae) is immunologically related to carboxypeptidases (10) and shows significant homology to various carboxypeptidases at the amino acid level. A potential catalytic triad has been identified that includes a serine, an aspartate, and a histidine (10).
␣-Hydroxynitrile lyase from Hevea brasiliensis (tropical rubber tree) is a class II enzyme and has been purified to homogeneity by Schall et al. 2 The subunit molecular mass of the unglycosylated enzyme is 30 kDa, and the enzyme activity is cofactor-independent. In low salt buffer solutions the enzyme is active as a homodimer. In contrast to other hydroxynitrile lyases, Hnl from H. brasiliensis has specificity for both aliphatic and aromatic (S)-cyanohydrins (11). 3 Here we report on the molecular cloning and sequence of a full-length cDNA of Hnl from Hevea brasiliensis. The cDNA was expressed in E. coli and Saccharomyces cerevisiae, and functional enzyme was produced and isolated from both systems. Using inhibitors and in vitro mutagenesis, we show that amino acid Cys 81 is required for enzymatic function.
Hydroxynitrile Lyase Preparation-Young leaves of H. brasiliensis were frozen in liquid nitrogen and crushed to powder. Proteins were extracted by stirring 2 g of powder in 5 ml of extraction buffer (20 mM potassium phosphate, pH 6.0) for 1 h on ice. Insoluble material was removed by centrifugation at 12,000 ϫ g for 10 min. The supernatant was desalted on Econo-Pac 10 DG columns (Bio-Rad) and stored at Ϫ20°C.
Assay of Hnl Activity-Enzyme activity was determined by following the formation of benzaldehyde from 3.8 mM racemic mandelonitrile in assay buffer (50 mM sodium citrate buffer, pH 5.0). The increase of benzaldehyde absorbance at 280 nm was monitored at 25°C over 5 min, which was in the linear range of the assay. For inhibition studies (inhibitors used are listed in Table II) 1 ml of enzyme extract in assay buffer was incubated with 1 mM inhibitor for 30 min at room temperature. 25-50 l of the mixture was assayed for activity.
Preparation of Rabbit Polyclonal Anti-Hnl Antiserum-Hnl was purified to homogeneity from Hevea brasiliensis leaves 2 and used for induction of anti-Hnl antibodies in rabbit applying standard immunization techniques (14). Titer and monospecificity of obtained anti-Hnl antiserum were tested by Western blotting techniques (14), using protein extracts from Hevea brasiliensis.
DNA and RNA Manipulations-DNA manipulations, plasmid transformation into E. coli, and retrieval of DNA from E. coli were performed following standard procedures (14). Plant genomic DNA was isolated by the method of Rogers and Reichardt as described in Ausubel et al. (14). Total RNA was prepared from fresh leaves of H. brasiliensis (provided by the Botanical Garden of the Karl-Franzens Universitä t, Graz) following a guanidinium/cesium chloride extraction method (15). Poly(A) ϩ RNA was enriched on oligo-dT cellulose (16) using the mRNA Separator Kit (Clontech Laboratories Inc., Palo Alto, CA). RNA was separated under denaturing conditions on 1% formaldehyde agarose gels in a MOPS buffer system (20 mM 3-(N-morpholino)propanesulfonic acid, 1 mM EDTA; pH 7.4). Hybridization of RNA or DNA transferred to nylon filters was performed as described (14) using a nonradioactive digoxigenin-probe labeling system (Boehringer Mannheim). An oriented cDNA library was constructed by employing a Uni-ZAP XR II phage vector and Gigapack Gold II packaging system, according to the supplier's instructions (Stratagene Cloning Systems, La Jolla, CA).
Immunoscreening of the cDNA Library-Phage from the library were plated at a density of approximately 10 3 plaque-forming units on NZY plates (0.5% NaCl, 0.2% MgSO 4 ⅐H 2 O, 0.5% yeast extract, 1% NZ amine, 2% agar; Ø 150 mm) using E. coli XL1-Blue host cells (preculture grown to A 600 ϭ 1.0). After cultivating phage for 8 -10 h, plates were overlaid with a nylon filter soaked in 10 mM isopropyl-1-thio-␤-D-galactopyranoside, and expression from the lac promoter was induced by incubation for 3 h. Filters were removed from the plates, blocked, and incubated 3 N. Klempier, personal communication.
with anti-Hnl primary antibody and anti-rabbit secondary antibody following the protocol of the Western Light Chemoluminescent Detection System (Tropix Inc., Bedford, MA). Anti-Hnl antiserum was diluted in 1 ϫ PBS (0.65 M NaPO 4 , 0.68 M NaCl, pH 7.4) containing 0.1% Tween 20 and 0.1% I-Block (Tropix Inc.). Specifically bound antibodies were visualized using alkaline phosphatase and the chromogenic substrate nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate, 4-toluidinium salt (Boehringer Mannheim). Positive phage clones were converted to plasmid derivatives of pBluescript SK Ϫ by in vivo excision (Stratagene Cloning Systems, La Jolla, CA). Heterologous proteins were characterized by SDS-polyacrylamide gel electrophoresis and Western blot analysis of E. coli extracts, leading to the identification of clone pHNL-100. Sequence Analysis-DNA-sequencing was performed by the chain termination method (17) using an ABI 373A automated sequencer and the Dye Deoxy Terminator Sequencing Kit (Applied Biosystems Inc.). The sequence of N-terminal and internal peptide fragments generated by proteolytic cleavage of purified Hnl with immobilized Lys-C was determined at a commercial protein sequencing facility (TopLab GmbH, FRG).
Comparison to nucleic acid sequence data bases (GenBank TM release 84.0 and EMBL release 39.0, and releases since) and protein data bases (Swiss-Prot release 30, PIR release 41.10, Genpept release 85 and Brookhaven Protein Data Base release April 1994), respectively, was performed via an electronic mail implementation of FASTA (18) at EMBL Mail Services and BLAST at the National Center of Biotechnology Information (Bethesda, MD) (19). Secondary structure, solvent accessibility, and membrane-spanning regions were determined using Predict Protein at EMBL (20) via electronic mail service. Membrane-spanning regions were predicted according to algorithms by Rao and Argos (21) and Klein (22). Sequence computation was done with the GCG package (GCG Program Manual for the Wisconsin Package, version 8, September 1994, Genetics Computer Group, Madison, WI).
Cloning of the 5Ј-End of the hnl Gene-Total phage DNA of the H. brasiliensis cDNA library was prepared as described by Grossberger (23). The 5Ј-end of hnl was cloned by two-step PCR. The following primers were used: primer 1, 5Ј-CCTCCAAGAACGTCAACAAG-3Ј; primer 2, 5Ј-CATCAAATGAGCCAATCTCC-3Ј with antisense orientation; vector-specific primer T3, 5Ј-AATTAACCCTCACTAAAGGG-3Ј. PCR was performed in a total volume of 20 l (25 pmol of primer, 0.2 mM dNTP, 1 ϫ reaction buffer (10 mM Tris, 50 mM KCl, 1.5 mM MgCl 2 , 0.001% gelatin, pH 8.8) and 2 units of Taq polymerase (Stratagene Cloning Systems, La Jolla, CA)) for 30 cycles of 1 min at 94°C, 1 min at 52°C, and 1 min at 72°C. The first linear amplification reaction contained primer 1 and 40 ng of DNA. In the second reaction primers 2 and T3 and 1 ⁄10 volume of reaction 1 as DNA template were used. The PCR products were purified and subcloned into plasmid pHNL-100, to yield pHNL-101.
A hnl fragment flanked by suitable restriction sites was constructed by replacing the 5Ј-and 3Ј-ends with PCR-generated fragments. Plasmid pHNL-101 containing the full-length hnl cDNA was used as the template. The following primers were used: 5Ј-end sense primer (containing EcoRI and NcoI sites), GGAATTCCATGGCATTCGCTCATTTT; 5Ј-end antisense primer, CATCAAATGAGCCAATCTCC; 3Ј-end sense primer, CACGCTTCTCTGAGGGAAAAT; 3Ј-end antisense primer (containing XhoI and HindIII sites), CCGCTCGAGAAGCTTCAAAGAAGT-CAATTATAG. The PCR fragments were gel-purified, cut with proper restriction enzymes, and used to replace the respective fragments in pHNL-101, resulting in plasmid pHNL-103. The correct nucleotide sequence was confirmed by sequence analysis.
Plasmid pHNL-103 was modified at the 3Ј end of the cDNA insert to introduce EcoRI and BamHI restriction sites. The HindIII-linearized pHNL-103 was ligated with a double-stranded adaptor (AGCTTGAAT-TCGATCC; AGCTGGATCCGAATTCA) obtaining construct pHNL-104. The presence of the adaptor sequence was confirmed by sequencing.
Site-directed Mutagenesis-Cys 81 (TGT) was changed to Ser (TCT) with PCR, using plasmid pHNL-104 as DNA template and the following primers: gene-specific mutant primer in antisense orientation, 5Ј-CAG-CAATTGCTATATTGAGTCCTCCAGAGCTCTCGC-3Ј, containing a MunI restriction site, and vector-specific primer, T3, 5Ј-AATTAACCCT-CACTAAAGGG-3Ј. The PCR conditions were 25 pmol of primer, 0.2 mM dNTP, 1 ϫ reaction buffer (10 mM Tris, 50 mM KCl, 1.5 mM MgCl 2 , 0.001% gelatin, pH 8.8) and 2 units of Taq polymerase (Stratagene, La Jolla, CA) for 30 cycles consisting of 1 min at 94°C, 1 min at 52°C, and 1 min at 72°C in a reaction volume of 20 l. The synthesized mutant DNA was cut with appropriate restriction enzymes, purified from an agarose gel and subcloned to obtain pHNL-105. The correct base replacement was confirmed by sequencing.
Construction of E. coli Expression Plasmid pHNL-200 and pHNL-201-The NcoI-HindIII fragments containing the entire coding region of the hnl wild-type gene (pHNL-103) and hnl mutant gene (pHNL-105) were subcloned into the respective sites of expression vector pSE420 to form plasmids pHNL-200 and pHNL-201.

Construction of S. cerevisiae Expression Plasmid Containing Wildtype and Mutant
Hnl cDNA-The hnl wild-type cDNA fragment (pHNL-104) and the hnl mutant cDNA fragment (pHNL-105) were subcloned as BamHI fragments into the unique BglII cloning site of pMA91 (13). Proper orientation was confirmed by partial sequencing of the resulting expression plasmids, pHNL-300, and the Cys 81 3 Ser mutant variant, pHNL-304.
Heterologous Expression in E. coli-E. coli strains XL1-Blue or TOP10Ј containing plasmid pHNL-200 were grown with great aeration (250 rpm) in 100 ml of 2 ϫ YT (10 g/liter NaCl, 10 g/liter yeast extract, 16 g/liter Bacto tryptone) supplemented with 100 mg/liter ampicillin at 37°C. Protein expression was induced with 1 mM isopropyl-1-thio-␤-Dgalactopyranoside over 3 h at various temperatures (37, 28, and 24°C). Cells were harvested by centrifugation, suspended in 4 ml of breaking buffer (50 mM potassium phosphate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, pH 7.4), and sonicated for 3 min at 45 watts on ice. Debris and insoluble fractions (inclusion bodies) were removed by centrifugation at 27,000 ϫ g for 15 min at 4°C. The supernatant contained soluble Hnl. Refolding of Hnl recovered from the insoluble fraction was performed according to Martson (24). The pellet was washed once with 2 M urea, 0.1 M Tris, pH 8.5, and resolubilized in 3.5 M urea, 0.1 M Tris, pH 8.5. The solution was dialyzed against breaking buffer containing 0.5 M urea followed by 2 ϫ dialysis against breaking buffer alone.
Production of Recombinant Hnl Protein in Saccharomyces cerevisiae-Yeast strains containing the Hnl expression plasmid pHNL-300 and its Cys 81 3 Ser mutant variant, pHNL-304, were grown for 16 h in 100 ml of YEPD (A 600 ϳ 12) and in leucine-free defined medium (A 600 ϳ 5) in baffled flasks at 30°C. Yeast cells were harvested by centrifugation and suspended in breaking buffer (50 mM K-PO 4 , 1 mM EDTA, pH 7.4). Cytosolic protein preparations were obtained by the "glass bead method" (14). Wild-type and mutant Hnl proteins were purified by one step ion exchange fast protein liquid chromatography on a Resource Q column (Pharmacia Biotech). The column was equilibrated with loading buffer (10 mM histidine/H 2 SO 4 , pH 6.7), and Hnl was eluted with a linear gradient of loading buffer/loading buffer plus 0.3 M (NH 4 ) 2 SO 4 . The protein was collected at a concentration of approximately 0.12 M (NH 4 ) 2 SO 4 and concentrated and desalted with an Omegacell TM ultrafiltration unit (Filtron Technology Corp., Northborough, MA).
Miscellaneous Procedures-Proteins were separated by polyacrylamide gel electrophoresis according to King and Laemmli (25) using 12.5% SDS-polyacrylamide gels or 7.5% native polyacrylamide gels. Isoelectric focusing was carried out with a PhastSystem (Pharmacia) using a pH 3-9 isoelectric focusing gel and pI 3.6 -9.3 isoelectric focusing markers. For Western analysis proteins were blotted onto nylon filters and detected as described above or with horseradish peroxidase and chemoluminescent substrates (14). Protein concentration was determined according to Lowry (26) using bovine serum albumin as the standard.

RESULTS
Cloning of Full-length hnl cDNA from H. brasiliensis-Young leaves of a 10-year-old H. brasiliensis tree containing hydroxynitrile lyase specific activity at about 0.4 units/mg of protein extract were the source of RNA used to construct the cDNA library. From 5 g of poly(A) ϩ RNA a cDNA library with 2 ϫ 10 6 plaque-forming units per packaging reaction was obtained and amplified. The library contained more than 99% recombinants with an average insert size of 1000 base pairs. About 100,000 plaques were screened with rabbit anti-Hnl anti-serum as described under "Materials and Methods." 12 positive phage clones were picked and converted into plasmids. The hybrid proteins expressed in E. coli were subjected to Western blot analysis, and extracts were assayed for Hnl activity. The clone harboring plasmid pHNL-100 expressed Hnl specific activity of 0.035 units/mg cytosolic protein and contained a 33-kDa fusion protein strongly reacting with anti-Hnl antiserum. The cDNA was sequenced, and an open reading frame of 255 amino acid residues downstream of the lacZ part was identified. Partial sequence analysis suggested that other immunoreactive clones may represent H. brasiliensis homo- logues of Zea maize 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, ferredoxin-NADP ϩ reductase from Pisum sativum and seduheptulose-1,7-bisphosphate from Tritium aestivum (more than 80% homology over at least 120 amino acids; data not shown).
The full-length hnl cDNA was cloned by PCR as described under "Materials and Methods" and sequenced. Among the four characterized cDNA clones, which varied by 4 nucleotides at their 5Ј ends, the longest cDNA clone extended the first isolate by 47 base pairs at the 5Ј-end. Two in-frame stop codons upstream of the open reading frame indicate that the complete hnl cDNA was isolated. Nucleotide sequence and the predicted amino acid sequence are shown in Fig. 1. The size of the cDNA of 1150 base pairs corresponds to the size of the hnl transcript of 1130 Ϯ 30 nucleotides, as determined by Northern blot analysis (Fig. 2). The low frequency of immunoreactive clones in the expression library as well as the weak signal detected with 10 g of poly(A) ϩ RNA suggest that hnl is only weakly expressed in leaves of H. brasiliensis that were used for our studies. Genomic DNA cleaved with different restriction endonucleases and probed with the isolated cDNA indicated that hnl is a unique gene with at least one intron that includes an EcoRI site (Fig. 3).
Characterization of the hnl cDNA-The open reading frame contained in the hnl cDNA encodes a protein with 257 amino acids with a predicted molecular mass of 29,227 Da. This prediction is in agreement with the plant protein subunit molecular weight of 30 kDa as determined by SDS-polyacrylamide gel electrophoresis. 2 Proof that the cloned hnl gene encodes the Hnl enzyme was obtained by protein sequence analysis of proteolytic fragments of Hnl purified from leaves of H. brasiliensis. Experimentally determined protein sequences are underlined in Fig. 1. The DNA sequence around the putative start ATG fulfills the known criteria for translation initiation sites of plant genes (27). The N terminus of the purified protein was blocked and therefore was not accessible to sequencing. No consensus sequences for N-glycosylation sites are present, supporting earlier findings that Hnl is unglycosylated. 2 Functional expression of H. brasiliensis Hydroxynitrile Lyase in E. coli and S. cerevisiae-hnl was expressed in E. coli strains XL1-Blue and TOP10Ј under the control of the trc promoter. The Hnl enzyme activities in various preparations are summarized in Table III. Cytosolic Hnl activity depended on the E. coli strain and the temperature during induction with isopropyl-1thio-␤-D-galactopyranoside. An inverse correlation between induction rate and solubility of the heterologous protein was observed. Insoluble fractions were devoid of active protein but contained inactive Hnl that appeared to be cross-linked by intra-and intermolecular disulfide bridges. A broad smear of protein in the high molecular weight range was observed on nonreducing SDS-polyacrylamide gels in addition to the 30-kDa Hnl subunit (data not shown). Thus, high level protein expression may lead to the formation of inclusion bodies. After solubilization and refolding of protein from the insoluble fraction less than 5% of Hnl was recovered in an active form (Fig.  4). However, the presence of active enzyme in the heterologous system provides further definitive proof that the cloned gene encodes Hnl.
Hnl was also expressed in S. cerevisiae. In this host system, Hnl could be obtained in high yield in a soluble form of up to 20% of the cytosolic protein. Recombinant Hnl purified from S. cerevisiae had a specific activity of 22 Ϯ 3 IU/mg, which is similar to the specific activity of 18 Ϯ 3 for the enzyme that was isolated from H. brasiliensis leaves (Table IV).
Protein Sequence Analysis-(S)-Hydroxynitrile lyase from H. brasiliensis shares 74% identity with Hnl from another Euphorbiaceae species, Manihot esculenta (28). Both enzymes are members of the class of non-FAD hydroxynitrile lyases. 35% identity with two proteins of unknown function from Oryza sativa (29) suggests functional or structural relationships of these proteins to Hnl. No significant homology to (R)-hydroxynitrile lyase (FAD) from Prunus serotia (7) or (S)-Hnl (non-FAD) from S. bicolor (10) is evident.
Hydroxynitrile lyases from H. brasiliensis and M. esculenta, and also the two Oryza proteins of unidentified function, share weak homology with the C termini of soluble mammalian epoxide hydrolases (30). These enzymes have been classified as new members of the ␣/␤-hydrolase fold protein family, containing Asp-Asp-His as the catalytic triad residues (31). Despite low primary sequence similarity members of this protein fam-  ily show a similar three-dimensional fold and conserved sequence order for the catalytic triad residues (32). Based on sequence homology to soluble mammalian epoxide hydrolases with its postulated catalytic triad, a potential catalytic triad Ser-Asp-His is suggested for the H. brasiliensis and M. esculenta enzymes (Fig. 5). (S)-Hydroxynitrile lyase from S. bicolor (10) has significant homology to various serine-carboxypeptidases, further supporting the notion of an active site serine residue. However, the same region around Ser 80 in the H. brasiliensis enzyme also contains the aldehyde dehydrogenase motif, indicating a potential catalytic role of Cys 81 . Hnl of several species of Prunus (8,33,34) and of Ximenia americana (35) and Sorghum vulgare (36) are inhibited by thiol reagents. Also, a cysteine residue can be chemically modified with pseudosubstrates such as ␣,␤-unsaturated propiophenones. Furthermore, the putative active center of Hnl from H. brasiliensis shares significant homology to the lipase and carboxypeptidase active site patterns (Fig. 6). These patterns contain serine or cysteine as the catalytically active residues, which are both present in Hnl.
Determination of the Active Site of Hnl-In order to further investigate the potential catalytic function of Cys 81 we replaced it with serine. The mutated protein, Hnl-Ser 81 , was expressed in E. coli and S. cerevisiae. Hnl activity in cytosolic protein extracts was completely abolished in E. coli (Table III), suggesting that Cys 81 is essential for catalytic activity. Since Hnl expression in E. coli resulted in rather low levels of active enzyme, we also expressed wild-type and mutated Hnl in S. cerevisiae. High expression levels (20% of cytosolic fraction) were achieved in this system. Mutated Hnl, Hnl-Ser 81 , was expressed at the same level as the wild-type enzyme. No differences in the electrophoretic mobility between the mutant and wild-type protein were observed on native polyacrylamide (Fig. 7) and isoelectric focusing gels (data not shown), indicating that the amino acid substitution did not cause major structural alterations. As can be seen from the data summarized in Table IV, the specific activity of the purified Hnl-Ser 81 protein was less than 2% of the specific activity determined for the wild-type Hnl protein. This further supports our view of a possible involvement of Cys 81 in Hnl catalysis.
Inhibition studies (Table II)   might be in a deep pocket not accessible to larger molecules, as was described for other proteins (37). However, participation of Ser 80 in the catalytic activity cannot be excluded at present, since serine-specific inhibitors like (4-amidinophenyl)-methanesulfonyl fluoride may be too bulky to access the active site.

DISCUSSION
Hydroxynitrile lyases have attracted great interest in basic plant biology as well as in biocatalytic applications. Here we report for the first time the cloning and molecular characterization of a class II enzyme from H. brasiliensis that accepts both aliphatic and aromatic (S)-cyanohydrins as substrates (11). Hnl from H. brasiliensis consists of a single polypeptide of 257 amino acids that does not appear to require extensive post-translational modification such as N-glycosylation for activity. This is in marked contrast to (R)-hydroxynitrile lyases, which are highly glycosylated, and to (S)-hydroxynitrile lyase from S. bicolor, a glycoprotein that requires post-translational proteolytic processing for activity (10). The N terminus of the H. brasiliensis protein appears to be blocked; by analogy to the M. esculenta enzyme and by applying rules for the N-terminal structure of intracellular proteins in eukaryotes (38) the Nterminal amino acid should be an acetylated alanine. The Nterminal 20 amino acid residues are hydrophobic and are predicted to adopt a ␤-fold conformation. We suggest that this region of the protein may act as a dimerization signal rather than as a membrane anchor, since we found that the soluble protein exists as a homodimer in low salt buffer. 2 Protein sequence analysis revealed high overall homology of the H. brasiliensis enzyme to Hnl from M. esculenta and moderate but significant homology to two proteins from rice (O. sativa). One of the rice proteins is induced after infiltration of leaves with Pseudomonas syringae, leading to speculation that it may be involved in a resistance response. Although sequence comparison suggested a lipase function (29), based on the structural considerations derived from the present study it is tempting to speculate that the rice protein may function as a hydroxynitrile lyase. 4 Although class I and class II hydroxynitrile lyases catalyze similar reactions, the overall molecular structure and organization of the enzymes and their active sites appear to be completely different. Furthermore, within the class II enzymes two subgroups can be proposed, one comprising the glycosylated, carboxypeptidase-like Sorghum type and the other a nonglycosylated, unprocessed Euphorbiaceae type. Enzymes of the two subgroups indeed differ in their active site architecture. Active site residue Cys 81 , which is essential for the H. brasiliensis enzyme, is absent in the proposed active site region of the S. bicolor enzyme (10). Inhibition of Sorghum sp. Hnl by mercury chloride (36) may be due to a disintegration of disulfide bridges required to establish heterotetrameric structure rather than inactivation of an active site cysteine residue. Lack of sequence homology among Hnls from different plant families indicates that these enzymes may have evolved convergently from different precursor structures. Thus, nature has adjusted enzyme activities for the retroaddition of HCN to a carbonyl group most likely resulting in slightly different mechanistic solutions. This may also explain the heterogeneous substrate specificities and stereoselectivities of the various Hnls. Hydroxynitrile lyase from H. brasiliensis was expressed in E. coli despite its rather low codon bias index for this host. Under the conditions of our expression system Hnl protein aggregated and formed inclusion bodies that could not be efficiently refolded into an active form by standard methods. In contrast, substantial overexpression of active Hnl could be achieved in the yeast S. cerevisiae, which thus appears to be a much more efficient host system for heterologous Hnl production.