(Received for publication, September 6, 1996, and in revised form, November 13, 1996)
From the Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
Acetone cyanohydrin lyase from Linum
usitatissimum is a hydroxynitrile lyase (HNL) which is involved
in the catabolism of cyanogenic glycosides in young seedlings of flax.
We have isolated a full-length cDNA clone encoding L. usitatissimum HNL (LuHNL) from a cDNA expression library by
immunoscreening. LuHNL cDNA was expressed in Escherichia
coli and isolated from the respective soluble fraction in an
active form which was biochemically indistinguishable from the natural
enzyme. An open reading frame of 1266 base pairs encodes for a protein
of 45,780 kDa. The derived amino acid sequence shows no overall
homologies to the to date cloned HNLs, but has significant similarities
to members of the alcohol dehydrogenase (ADH) family of enzymes. In
particular, the cysteine and histidine residues responsible for
coordination of an active site Zn2+ and a second
structurally important Zn2+ in alcohol dehydrogenases are
conserved. Nevertheless, we found neither alcohol dehydrogenase
activity in LuHNL nor HNL activity in ADH. Moreover, well known
inhibitors of ADHs, which interfere with the coordination of the active
site Zn2+, fail to affect HNL activity of LuHNL, suggesting
principally different mechanisms of cyanohydrin cleavage and alcohol
oxidation. Interestingly, LuHNL like ADH and Prunus
serotina (PsHNL) possesses an ADP-binding unit motif,
pointing to the possibility that the non-flavoprotein PsHNL and the
flavoprotein LuHNL have developed from two independent lines of
evolution of a common ancestor with an ADP-binding
unit.
Hydroxynitrile lyases (HNLs)1 catalyze
the decomposition of cyanohydrins (-hydroxynitriles) into the
corresponding aldehyde or ketone and cyanide (1). All HNLs described so
far are found in cyanogenic plants. In these plants, HNLs are involved
in the catabolization of cyanogenic glycosides during cyanogenesis or in the metabolization of these compounds during seedling development (1-4). In the presence of high concentrations of HCN and aldehydes or
ketones, HNLs can be used as biocatalysts for the stereoselective synthesis of a wide array of cyanohydrins, important building blocks in
the pharmaceutical and fine chemical industries
(5).
In recent years the HNLs from Prunus serotina (PsHNL) (6),
Sorghum bicolor (SbHNL) (7), Manihot esculenta
(MeHNL) (8), and Hevea brasiliensis (HbHNL) (9) have been
molecularly cloned. Analysis of the cDNA-derived amino acid
sequences revealed that these enzymes belong to three different classes
of HNL. While MeHNL and HbHNL share 74% identity (9), no sequence
homologies can be found to PsHNL or SbHNL. The lack of sequence
homologies between the cloned HNLs correlates with the fundamental
differences between these enzymes with regard to molecular weight,
subunit composition, glycosylation, FAD content, and substrate
specificity.2 The flavoprotein PsHNL has moderate
homologies to various other flavoproteins, especially to various types
of dehydrogenases and oxidases (6). In particular, a stretch of 27 amino acid residues near the N terminus of PsHNL fulfill Wierenga's
rule for an ADP-binding unit (6), while MeHNL and HbHNL show
homologies to two proteins of unknown function from rice (9). However,
the most intriguing homologies are found for SbHNL, namely, that this
HNL possesses up to 50% homology over the whole sequence to serine carboxypeptidases, which belong to the structurally well investigated group of
/
hydrolase fold enzymes (5, 7). In particular, sites
critical for function and structural integrity of serine carboxypeptidases are conserved, suggesting that SbHNL is also a
/
hydrolase fold enzyme. All
/
hydrolase fold enzymes have a "nucleophile-histidine-acid" catalytic triad found in common with
the subtilisin and chymotrypsin class of serine proteases (10). In all
these enzymes, the nucleophile is part of the consensus motif
Gly-X-Ser/Cys-X-Gly/Ala-Gly/Ala (10). There is
functional evidence by site-directed mutagenesis for the use of a
catalytic triad by MeHNL and HbHNL, as well (9, 11). Moreover, the order of the catalytic triad residues in primary sequence suggests that
these HNLs also belong to the
/
hydrolase fold group of enzymes
despite having no sequence homologies to SbHNL (11).
Here we describe the molecular cloning of LuHNL, which, like MeHNL and
HbHNL, has acetone cyanohydrin as its natural substrate. However, in
contrast to these presumed /
hydrolase fold enzymes, LuHNL
was found to be structurally related to the alcohol dehydrogenase class
of enzymes. In particular, amino acid residues of ADHs important for
structural integrity or coordinating Zn2+ are conserved in
LuHNL. However, despite having all the conserved residues responsible
for Zn2+ binding, LuHNL neither exerts ADH activity
nor is inhibited by reagents interfering with Zn2+
coordination in liver ADH.
Seeds of Linum usitatissimum L. were obtained from Frank AG (Herrenberg, Germany). Seeds were
germinated for 5-10 days at room temperature under normal daylight
conditions. The cotyledons of the seedling were cut and stored at
20 °C or used immediately for enzyme purification and RNA
isolation.
Except where noted, chemicals were purchased from Sigma (Deisenhofen, Germany). Chromatography resins, AutoRead sequencing kit, and cDNA synthesis kit were obtained from Pharmacia LKB Biotechnology Inc. (Freiburg, Germany). Lambda ZAP DNA was from Stratagene, and the bicinchoninic acid protein assay kit was from Pierce.
Enzyme AssaysThe activity of LuHNL was measured as described by Selmar et al. (12). The amount of HNL which decomposes 1 µmol of acetone cyanohydrin in 1 min under the conditions described in Selmar et al. (12) is defined as 1 unit. Total protein for calculation of specific activities was determined with the bicinchoninic acid protein assay reagent from Pierce according to the manufacturer's recommendations. Enzyme assays for liver alcohol dehydrogenase (Sigma) were essentially performed as described by Dunn and Bernhard (13). For inhibition studies, enzymes were incubated in the appropriate reaction buffer containing the respective reagent for 30 min at room temperature. The remaining activity was then determined as described above.
Purification of LuHNL and ImmunizationPurification of LuHNL from flax seedlings and the immunization procedure were performed as described previously (14). Purification of recombinant LuHNL was performed as follows. E. coli cells expressing LuHNL were harvested by centrifugation, resuspended in binding buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl), and disrupted by sonication on ice. The supernatant was cleared by centrifugation and applied on a 1-ml Ni-nitrilotriacetic acid Superflow column (Quiagen, Minden, Germany). After washing with 10 ml of binding buffer, bound proteins were eluted with a 30-ml gradient of 0-0.5 M imidazole in binding buffer.
Generation and Screening of a Lambda ZAP II cDNA LibraryTotal RNA was isolated from equal amounts of 5-, 7-, and 10-day-old seedlings of L. usitatissimum according to the guanidine chloride procedure described by Logeman et al. (15). Poly(A)+-enriched RNA was obtained using Oligotex(dT) particles (Quiagen, Minden, Germany). Oligo(dT)-primed cDNA was obtained with a cDNA synthesis kit (Pharmacia) according to the manufacturer's recommendations and cloned in lambda ZAP II (Stratagene). A total of 1 × 105 recombinants were screened with polyclonal antisera (1:1000) against LuHNL as described by Young and Davis (16). Positive clones were visualized by detection of the formed complex of anti-LuHNL antibodies and anti-mouse (Ig) antibodies conjugated with alkaline phosphatase (0.4 µg/ml; Dianova, Hamburg, Germany). Detection was performed with a combination of 0.01% 5-bromo-4-chloro-3-indolyl phosphate and 0.02% nitro blue tetrazolium complemented with 4 mM MgCl2 as a substrate. After in vivo excision of the pBluescript plasmid, the cDNAs encoded by the positive clones were further characterized.
DNA Sequencing and AnalysisSequencing of double-stranded DNA templates was achieved by a modified chain-termination method (17) using T7 DNA polymerase and the ALF express DNA analysis system (Pharmacia). For priming, T3 and T7 primer or specific oligonucleotides based on the preceding sequences were used. Obtained DNA and amino acid sequences were compiled and analyzed using the HUSAR (Heidelberg UNIX Sequence Resources) software.
SDS-PAGE and ImmunoblottingProteins were separated on 15% (w/v) polyacrylamide gels according to Laemmli (18) and either silver stained according to Blum et al. (19) or transferred to nitrocellulose as described by Towbin et al. (20). The immunoblots were further handled as described elsewhere (14).
A size selected (800-2500 base pairs) cDNA
expression library in lambda ZAP comprising 5 × 105
plaque-forming units per packing reaction (85% recombinants) was used
without further amplification for screening with anti-LuHNL antisera.
About 100,000 plaques were screened as described under "Experimental
Procedures." Three positive clones, containing inserts of 1.3, 1.4, and 1.5 kilobase pairs in length were identified, converted into
plasmid and used for further analysis. Both strands of the cDNAs
were sequenced, and a common open reading frame of 422 amino acid
residues downstream of the lacZ part was identified. The open reading
frame encodes for a protein with a predicted molecular mass of 45,780 which is in good accordance with the molecular mass of 42,000 estimated
for purified LuHNL by SDS-PAGE (21). In particular, the open reading
frame contained sequences near the start methionine corresponding to
the N-terminal sequence of LuHNL determined by Albrecht et
al. (22) by Edman degradation. Two potential polyadenylation
signals (AATAAA) occur at 1429 and 1507. The nucleotide sequence and
the predicted amino acid sequence are shown in Fig.
1.
Protein Sequence Analysis
Searching for homologies using
HUSAR and the TFASTA algorithms revealed that LuHNL shares up to 40%
homology over the whole molecule with members of the zinc-containing
alcohol dehydrogenase family of enzymes (Fig. 2). Of
great interest is the fact that there are no significant homologies of
LuHNL to the other HNLs cloned so far. Remarkably, residues which are
structurally or functionally important in alcohol dehydrogenases are
conserved (Fig. 2). Like LuHNL, alcohol dehydrogenases consist of two
identical subunits (23). For ADHs it has been shown by resolution of
the three-dimensional structure that each subunit is divided into two
domains separated by the deep active site cleft (23). In ADHs one of
these domains binds the coenzyme (coenzyme binding domain). The other
domain binds two zinc ions (catalytic domain), one of which is located
in the active-site pocket, whereas the other one is located on a lobe
outside the catalytic center (23). All residues responsible for
Zn2+ binding in ADHs are conserved in LuHNL (Fig.
2A). Three glycines (LuHNL: Gly84,
Gly95, and Gly104) of ADHs which are invariant
because of lack of space for a side chain in hydrophobic cores are also
conserved (Fig. 2A). What is more, analyzing the deduced
LuHNL amino acid sequence according to Wierenga's rules (24) revealed
a ADP-binding -unit motif comprising residues 219-248 (Fig.
2A). Based on analysis of several FAD- and NAD-binding
proteins, Wierenga et al. (24) have defined 11 residues
within a 29-31 amino acid motif, which are necessary to allow the
sequence folding into an ADP-binding
unit. The ADP-binding
unit motif of LuHNL, compared in Fig. 2B with those of other ADP-binding proteins, matched exactly with the consensus
sequence and is therefore very likely to prove to be folded as an
ADP-binding
unit (Fig. 2B). Nevertheless, LuHNL catalyzes no net oxidation or reduction, suggesting that this fold is
rather of structural than of catalytic importance. Taking into account
the above mentioned conservation of structurally important residues
between LuHNL and ADHs, we propose that the overall structure of LuHNL
is quite similar to that of ADHs.
Inhibition Studies
Given the complete conservation of cysteine and histidine residues required for coordination of the active Zn2+ in alcohol dehydrogenases, we proposed two questions. First, has LuHNL a side dehydrogenase activity or have ADHs a side HNL activity and second, are the above mentioned conserved residues functionally involved in LuHNL-catalyzed acetone cyanohydrin cleavage? As shown in Table I, we found no ADH activity with LuHNL in a standard ADH assay; likewise, we found no indication for hydroxynitrile lyase activity in a commercially available liver alcohol dehydrogenase preparation. As mentioned above, one of the two coordinated Zn2+ of each ADH subunit is located in the active site and functionally involved in catalysis. Therefore both o-phenanthroline, which forms complexes with Zn2+, and diethyl pyrocarbonate, which modifies histidine side chains, are potent inhibitors of ADH activity (23). Taking into account that the above mentioned residues responsible for Zn2+ coordination are conserved between LuHNL and ADHs, we inquired into a putative involvement in LuHNL-catalyzed cyanohydrin cleavage of these residues. Surprisingly, we found no influence of these inhibitors on LuHNL activity even after extended preincubation and at concentrations 10 to 20 times higher than those used for significant inhibition of ADH activity (Table I). The competitive inhibitor o-phenanthroline forms a ADH-Zn2+-o-phenanthroline complexes with the active-site Zn2+ of ADHs (25) leading to reversible inactivation of these enzyms. The lack of inhibition of HNL-activity by this agent therefore suggests that Zn2+ is not directly involved in LuHNL-catalyzed cyanohydrin cleveage.
|
However, a structural role of Zn2+ ions in LuHNL cannot be ruled out by these data. LuHNL was poorly inhibited by high concentrations (10 mM) of the serine-modifying reagent diisopropyl fluorophosphate (Table I), whereas HNLs having a catalytic triad were almost completely inhibited by this compound in the low millimolar range (7, 11). Hence, involvement of a serine residue in LuHNL catalysis is rather unlikely.
Functional Expression of LuHNLTo investigate the biochemical
properties of recombinant LuHN, we cloned LuHNL cDNA in the
inducible procaryotic expression vector pQE10 (Quiagen) and expressed
the protein in E. coli (Fig. 3). Recombinant
LuHNL was purified by affinity chromatography on a Ni-nitrilo triacetic
acid Superflow column with a linear gradient from 0 to 0.5 M imidazole in binding buffer. The purification process
yielded a pure enzyme preparation with a specific activity of 40 units/mg which is in accordance with the specific activity (34 units/mg) described for the natural enzyme. The recombinant enzyme
exhibited a Michaelis-Menten kinetic with a Km for
acetone cyanohydrin of 1.9 mM and
Vmax of 71 µmol/min/mg (Fig. 3), which again
matched well with the values published for the natural enzyme by Xu
et al. (21) (Km = 2.5 mM). In contrast to other HNLs, the substrate specificity of LuHNL has not yet
been studied in detail. Nevertheless, preliminary data of Albrecht
et al. (22) suggested that LuHNL acts preferentially on
aliphatic (R)-cyanohydrins. The molecular cloning of LuHNL described here should now allow the overexpression of this enzyme. A
more thorough investigation of the technological potential of this
enzyme with regard to its possible use as a biocatalyst in stereoselective synthesis of cyanohydrins should therefore be possible.
Phylogeny of Hydroxynitrile Lyases of Higher Plants
Previously, HNLs were divided into two fundamental classes
according to their FAD content. However, comparison of the amino acid
sequence of various recently cloned HNLs (PsHNL, SbHNL, MeHNL, HbHNL)
(6-9) revealed sequence homologies to other proteins, suggesting the
existence of at least three phylogenetically independent groups of
HNLs. One group formed by HbHNL and MeHNL, sharing 74% identity,
exerts significant homologies to two proteins of as yet unknown
function from rice (9). The other two groups are defined by SbHNL and
PsHNL, respectively. While sequence analysis of the SbHNL revealed
extensive homologies to serine carboxypeptidases, which belong to the
structurally well investigated group of /
hydrolase fold enzymes
(5, 7), the flavoprotein PsHNL shows moderate homologies to various
flavoproteins, especially to dehydrogenases and oxidases. Remarkably,
we found no sequence homologies of LuHNL with the HNLs from cassava
(MeHNL) and rubber tree (HbHNL), despite a common natural substrate
(acetone cyanohydrin). This lack of sequence homology is consistent
with the discrepancy in biochemical properties of these enzymes (Table
II). Therefore, we propose here that LuHNL defines a
fourth group of HNLs. LuHNL, like PsHNL, has an ADP-binding
unit motif matching strikingly with the conserved sequence defined by
Wierenga et al. (24) for this fold. Otherwise, there are no
overall sequence similarities between these two HNLs, suggesting that
they have evolved independently from two different lines of evolution
from an ancestoral ADP-binding
unit protein (Fig.
4). There is no indication of a phylogenetic relationship of LuHNL or PsHNL to the
/
hydrolase fold enzymes. Taking into account the lack of similarity in biochemical properties of
the, to date, noncloned HNLs from Ximenia americana (26) and
Phlebodium aureum (5) and the above discussed HNLs, it is
likely that additional, phylogenetically defined groups of HNLs exist
(Fig. 4).
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The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y09084[GenBank].
We gratefully acknowledge the technical assistance of Ute Emerich, Dr. Heiner Böttinger for immunization of mice, and Prof. Klaus Pfizenmaier for helpful discussion of the manuscript. We also thank James Parker for reading and correcting the manuscript.