(Received for publication, July 12, 1995; and in revised form, November 20, 1995)
From the
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) ()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)(+)-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 1) 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.(
)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) . (
)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
is
required for enzymatic function.
Yeast wild-type strain W303D (MATa/, leu2-3/112 leu2-3,112
his3-11,15/his3-11,15 ade2-1/ade2-1
ura3-1/ura3-1 trp1-1/trp1-1
can1-100/can1-100; (12) ) and plasmid pMA91
with a constitutive phosphoglycerate kinase promoter (13) were
used for protein expression in S. cerevisiae. Cells were grown
in YEPD (yeast extract (1 g/liter), Bacto peptone (2 g/liter), glucose
(3 g/liter)) or leucine-free defined medium (Difco yeast nitrogen base
without amino acids (6.7 g/liter), glucose (1 g/liter), adenine (20
mg/liter), arginine (20 mg/liter), histidine (20 mg/liter), lysine (230
mg/liter), methionine (20 mg/liter), threonine (300 mg/liter),
tryptophan (20 mg/liter), and uracil (40 mg/liter) to late exponential
phase.
Comparison to nucleic acid
sequence data bases (GenBank 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).
A hnl fragment flanked by suitable restriction sites was constructed by replacing the 5`- and 3`-ends with PCR-generated fragments. Plasmid pHNL-101 containing the full-length hnl cDNA was used as the template. The following primers were used: 5`-end sense primer (containing EcoRI and NcoI sites), GGAATTCCATGGCATTCGCTCATTTT; 5`-end antisense primer, CATCAAATGAGCCAATCTCC; 3`-end sense primer, CACGCTTCTCTGAGGGAAAAT; 3`-end antisense primer (containing XhoI and HindIII sites), CCGCTCGAGAAGCTTCAAAGAAGTCAATTATAG. 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 3` end of the cDNA insert to introduce EcoRI and BamHI restriction sites. The HindIII-linearized pHNL-103 was ligated with a double-stranded adaptor (AGCTTGAATTCGATCC; AGCTGGATCCGAATTCA) obtaining construct pHNL-104. The presence of the adaptor sequence was confirmed by sequencing.
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 5` ends, the longest cDNA clone extended the first isolate by
47 base pairs at the 5`-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 ±
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 µg
of poly(A) 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).
Figure 1:
Nucleotide sequence
of Hevea brasiliensis (S)-hydroxynitrile lyase cDNA
and derived amino acid sequence. Underlined sequences
correlate to data obtained by protein sequencing. A putative
polyadenylation signal is dotted underlined. The 5`-end of the
original cDNA clone derived from the immunoscreening is marked by a dot (). Cysteine 81, which is possibly involved in
catalytic action, is indicated by an asterisk.
Figure 2:
Northern blot analysis of Hnl transcript.
Poly (A) RNA isolated from young leaves of H.
brasiliensis was hybridized with digoxigenin-labeled hnl cDNA. hnl-specific mRNA of about 1130 nucleotides is
marked by an arrow.
Figure 3: Southern blot analysis of genomic DNA. Genomic DNA isolated from leaves of H. brasiliensis was cut with SacI (lane 1), HindIII (lane 2), and EcoRI (lane 3) and probed with digoxigenin-labeled hnl cDNA. HindIII did not cut to completion.
Figure 4: Western blot analysis of recombinant Hnl expressed in E. coli. Protein extracts of Hnl overexpressing E. coli strains were separated on a 7.5% native polyacrylamide gel and probed with anti-Hnl antiserum. Lane 1, soluble fraction of XL1-Blue harboring plasmid pSE420 (no insert); lane 2, soluble fraction of XL1-Blue harboring plasmid pHNL-200 (Hnl-cDNA); lane 3, partially refolded insoluble fraction of XL1-Blue/pHNL-200. The arrow marks the main conformation of Hnl in soluble fraction.
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 ± 3 IU/mg, which is similar to the specific activity of 18 ± 3 for the enzyme that was isolated from H. brasiliensis leaves (Table 4).
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 family 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
in the H. brasiliensis enzyme also contains the aldehyde
dehydrogenase motif, indicating a potential catalytic role of
Cys
. 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.
Figure 5:
Alignment of proteins with potential
structural or functional relationship. HNL_Hb and HNL_Me are
hydroxynitrile lyases from H. brasiliensis and M.
esculenta (GenBank and EMBL accession number Z29091),
respectively; Ospir7a and Ospir7b (EMBL accession numbers Z34270 and
Z34271) are amino acid sequences of unknown function translated from
two open reading frames identified in O. sativa. sEH_Hs is the
C-terminal part of the human soluble epoxide hydrolase (Swiss-Prot
accession number P34419; residues 251-548). The positions of the
catalytic triad suggested for mammalian epoxide hydrolase (28, 29) are marked by arrowheads. The bar marks the aldehyde dehydrogenase motif in H.
brasiliensis Hnl.
Figure 6: Alignment of putative active sites. Comparison of the active region motifs of lipases (LIP), aldehyde dehydrogenases (ADH), and carboxypeptidases (CPD) as obtained from the Prosite data base with the putative active site region of hydroxynitrile lyase (HNL) of H. brasiliensis. Amino acids in boldface type are involved in catalytic activity.
Figure 7: Heterologous expression of wild-type and mutant Hnl in S. cerevisiae. Soluble protein fractions of S. cerevisiae W303D transformed with pMA91 (no insert, lane 1), pHNL-300 (wild-type Hnl, lane 2), and pHNL-304 (mutant Hnl, lane 3) are shown. 10 µg of soluble protein/lane were separated by native polyacrylamide gel electrophoresis and stained with Coomassie Blue. Double bands of Hnl resolved by native polyacrylamide gel electrophoresis are probably due to partial N-terminal processing.
Inhibition studies (Table 2) support a potential
role of SH groups in catalysis. Hg(II) chloride and
parachloromercuribenzoate completely inactivated the enzyme, whereas
more bulky sulfhydryl reagents like N-ethylmaleimide or E-64 (N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatin)
had no effect on Hnl activity. These data suggest that the active
center might be in a deep pocket not accessible to larger molecules, as
was described for other proteins(37) . However, participation
of Ser 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.
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 N-terminal amino
acid should be an acetylated alanine. The N-terminal 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.
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. ()
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, 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U40402[GenBank].