An R-selective hydroxynitrile lyase from Arabidopsis thaliana with an α/β-hydrolase fold

Article abstract of DOI:10.1002/anie.200701455

Folding and selectivity: The noncyanogenic plant Arabidopsis thaliana contains a new hydroxynitrile lyase, which was cloned and characterized. This enzyme is readily available form a recombinant source, has a broad range of substrates, and enantioselectively transforms aliphatic and aromatic aldehydes as well as ketones into the corresponding R-cyanohydrins. (Chemical Equation Presented).

Full text of DOI:10.1002/anie.200701455

Angewandte
Chemie
DOI: 10.1002/anie.200701455
Enzyme Catalysis
An R-Selective Hydroxynitrile Lyase from Arabidopsis thaliana with
an a/b-Hydrolase Fold**
Jennifer Andexer, Jan von Langermann, Annett Mell, Marco Bocola, Udo Kragl,*
Thorsten Eggert,* and Martina Pohl*
Hydroxynitrile lyases (HNLs) catalyze the stereoselective
be easily and economically produced.These demands are
fulfilled by the currently available S-selective enzymes
HbHNL and MeHNL: they can be expressed in bacterial
hosts like Escherichia coli and accept a broad range of
À
formation of C C bonds between HCN and aldehydes or
ketones yielding chiral cyanohydrins, which are versatile
building blocks for the pharmaceutical and agrochemical
industries.[1] Among the most important cyanohdrins are
chiral a-hydroxy acids such as substituted mandelic
acids,[1e,f,2a] m-phenoxybenzaldehyde derivatives,[2c] and struc-
tures with additional aliphatic linkers between the aldehyde
moiety and aromatic ring which are useful for the synthesis of
“prils”.[2d] In nature HNLs catalyze the cleavage of cyanohy-
drins, known as cyanogenesis.The currently known HNLs can
be divided into two groups: R-selective enzymes evolved from
oxidoreductase ancestors, such as HNLs from various Rosa-
ceae[2] and from Linum usitatissimum,[3a] and S-selective
enzymes derived from hydrolases with an a/b-hydrolase
fold; these encompassing the enzymes from Hevea brasiliensis
(HbHNL),[3b] Manihot esculenta (MeHNL),[3c] and Sorghum
bicolor (SbHNL).[3d] Here we present the first exception to
this accepted rule with the first R-selective HNL containing
an a/b-hydrolase fold from the noncyanogenic plant Arabi-
dopsis thaliana (mouse-ear cress).
aromatic and aliphatic aldehydes as well as ketones.[4]
A
similar broad substrate range has been reported for the R-
selective HNLs isolated from some Prunus species (P. amyg-
dalus (PaHNL) and P. mume (PmHNL)).These biocatalysts
are either used as defatted seed meals or, in the case of
PaHNL (isoenzyme 5), are expressed in the yeast Pichia
pastoris.[2a,e]
Recently, several approaches were reported to identify
new HNLs for biocatalytic processes by screening different
cyanogenic plant extracts for HNL activity, yielding some new
enzyme sources.[5] Attempts to identify new enzymes based on
sequence similarities to known HNLs have not yet been
successful.[6,7]
Several sequences similar to MeHNL and HbHNL are
found in the genome of the noncyanogenic model plant
Arabidopsis thaliana.[7] In the course of our studies on
structure–function relationships of a/b-hydrolases we cloned
several genes encoding Arabidopsis proteins with high
sequence similarity to MeHNL and HbHNL and expressed
them in E. coli. Unexpectedly, one of them (gene bank entry:
AAN13041) shows high activity towards mandelonitrile and
catalyzes also the cleavage of some other cyanohydrins
derived from cyclohexanone and m-phenoxybenzaldehyde,
while acetaldehyde, propionaldehyde, and acetone cyanohy-
drin are poor substrates.[8]
Owing to the growing demand for chiral compounds like
cyanohydrins there is a strong motivation to identify new
stereoselective HNLs with a broad substrate range which can
[*] J. von Langermann, A. Mell, Prof. Dr. U. Kragl
Institute ofChemistry, University ofRostock
Albert-Einstein-Strasse 3a, 18059 Rostock (Germany)
Fax: (+49)381-498-6452
E-mail: udo.kragl@uni-rostock.de
A subsequent investigation of the cyanohydrin-forming
activity revealed that the new enzyme is highly R-selective
with a broad substrate range including various aromatic and
aliphatic aldehydes as well as ketones, which are converted to
R-cyanohydrins with good to excellent yields and mainly
excellent enantioselectivities (Table 1).[9] As can be seen, a
whole range of substituted benzaldehydes are converted with
excellent activity and enantioselectivity.There was no opti-
mization of the reaction time, but substrates such as 3, 4, and
6, which react even in the absence of the enzyme, gave
products with 99% ee indicating a high enzymatic activity
towards these substrates.To obtain complete conversion of
substrates with the more bulky substituents the reaction time
had to be increased slightly.It should be also noted that the
reaction was performed at pH 5.Lowering the pH could of
course suppress the nonenzymatic reaction even further.But
even at pH 5 the ee obtained is higher for o-chlorobenzalde-
hyde cyanohydrin than that in earlier studies with optimized
PaHNL[2a] or with the wild-type enzyme.[10] Subsequent
hydrolysis yields (R)-o-chloromandelic acid, which is a key
Homepage: http://www.chemie.uni-rostock.de/kragl
Dr. T. Eggert
evocatal GmbH
Merowingerplatz 1a, 40225 Düsseldorf(Germany)
E-mail: t.eggert@evocatal.com
Homepage: www.evocatal.com
J. Andexer, Dr. M. Pohl
Institute ofMolecular Enzyme Technology
Heinrich-Heine University ofDüsseldorf
52426 Jülich (Germany)
Fax: (+49)2461-612-940
E-mail: ma.pohl@fz-juelich.de
Homepage: www.iet.uni-duesseldorf.de
Dr. M. Bocola
University ofRegensburg
Department ofPhysical Biochemistry 2
Universitätsstrasse 31, 93053 Regensburg (Germany)
[**] The authors thank the group ofUte Höcker (University of
Düsseldorf, Botanik IV) for providing Arabidopsis cDNA and mRNA.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2007, 46, 8679 –8681
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
8679
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Communications
Table 1: Substrate range of AtHNL.[a]
Substrate
t [h]
Xenz[b] [%]
ee (R) [%]
Xnenz[b] [%]
R=H
R=o-F
R=o-Cl
R=o-Br
R=o-I
R=m-F
R=m-Cl
R=m-Br
R=m-I
1
2
3
4
5
6
7
8
9
2
2
2
6
3
2
3
6
6
>99
>99
>99
99
>99
>99
99
>99
99
99
14
17
26
42
26
22
7
98
>95
>99
>99
95
Scheme 1. AtHNL-catalyzed synthesis ofchiral cyanohydrins and exam-
ples ofcompounds obtained atfer subsequent reactions.
99
98
9
5
93
R=m-PhO
R=p-F
R=p-Cl
R=p-Br
R=p-I-
10
11
12
13
14
15
16
22
2
2
3
6
83
>95
>99
>99
>99
92
0
7
4
4
7
3
14
>99
>99
99
99
96
lectivity.In comparison, the enzyme is less active towards
aliphatic and aromatic ketones.
In order to rationalize similarities and differences con-
cerning the reaction mechanism and stereoselectivity of
AtHNL relative to the structurally similar, but S-selective
HbHNL and MeHNL, a homology model was created, based
on the crystal structures of HbHNL.[9,11] A comparison of
both structures suggests a typical catalytic triad consisting of
Ser81, Asp208, and His236 also in AtHNL (Figure 1).
R=p-OH-
R=p-OMe-
3
22
97
68
87
17
22
97
96
97
18
22
99
68
97
19
20
21
22
23
24
25
26
6
6
68
99
56
53
0
n.d.[c]
6
78
0
98
>95
n.d.[c]
–
22
3
0
22
6
0
48
2
95
2
22
3
–
0
[d]
94
–
76
27
22
7
n.d.
0
28
29
22
3
8
1
95
–
0
0
Figure 1. Overlay ofthe crystal structure of HbHNL (dark gray, thin
lines)[11] and the structural model of AtHNL (light gray, thick rods).
The catalytic triad (Ser/His/Asp) and the residues in contact with
bound mandelonitrile are shown: (R)-mandelonitrile in the AtHNL
model and (S)-mandelonitrile in the crystal structure of HbHNL
(1YB8). AtHNL reveals a specific binding pocket for (R)-mandelonitrile
between Leu129 and Ala13 which is blocked by Trp128 and Ile12 in
HbHNL.
[a] All conversions were performed in a two-phase system; conversion
(X) and enantiomeric excess (ee) were determined by gas chromatog-
raphy.[9] n.d.=not determined. [b] enz: enzymatic; nenz: nonenzymatic.
[c] Separation ofenantiomers by the Chiraldex capillary GC column (G-
PN-g-cyclodextrin, propionyl) was not possible. [d] Achiral product.
intermediate for the antithrombotic agent clopidogrel (30).
The cyanohydrin of 18 can be transferred to the correspond-
ing a-hydroxyester, which is a building block of ACE
inhibitors such as enalapril (31; Scheme 1).
The reaction of substrate 18 is somewhat less selective
than that of 17, indicating that the enzymatic reaction is
slower and therefore the reaction conditions, primarily the
pH, must be fine-tuned.Also an increasing chain length of the
aliphatic aldehydes reduces the activity but not the stereose-
These residues were exchanged by nonfunctional, but
sterically similar residues using site-directed mutagenesis, and
the resulting variants (Ser81Ala, Asp208Asn, His236Phe)
showed drastically impaired catalytic activity (< 2%), sup-
porting their catalytically important function.[9] A further
catalytically important residue (Lys236), which has been
identified in HbHNL,[11a] is replaced by Met237 in AtHNL.
To analyze the differences in stereoselectivity a structural
model of AtHNL with (R)-mandelonitrile bound to the active
8680 www.angewandte.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 8679 –8681
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Angewandte
Chemie
2001, 343, 547; f) G.Coppola, H.Schuster, a-Hydroxy Acids in
site was created based on the structure of HbHNL containing
(S)-mandelonitrile.[9,11] In comparison to HbHNL, two side
chains of the potential substrate-binding pocket in AtHNL
are exchanged.These are Trp128 and Cys13 in HbHNL,
which are replaced by Leu129 and Tyr14, respectively, in
AtHNL (Figure 1).The strict S selectivity of HbHNL can be
understood from the constructed model, since Trp128 and
Ile12 sterically hinder the binding of (R)-mandelonitrile.On
the other hand, it can be expected that the aromatic side
chains of Tyr14 and Phe82 might stabilize (R)-mandelonitrile
in the binding pocket of AtHNL.
In first experiments with an AtHNL variant (Tyr14Cys)
still exclusively (R)-mandelonitrile was produced, suggesting
that a single exchange is not sufficient to alter the stereose-
lectivity of AtHNL.Studies on a double mutant (Tyr14Cys/
Leu129Trp) and the crystal structure of the enzyme are in
progress.The homology model is not yet accurate enough to
explain the differences in activity or substrate selectivity as
discussed before.
We have described a novel R-specific HNL (E.C. 4.2.1.–)
from Arabidopsis thaliana and its application in biocatalytic
processes.The enzyme is a good alternative to currently
known R-selective HNLs, such as PaHNL,[2e] for the produc-
tion of R-cyanohydrins as it is readily available in technically
relevant amounts by overexpression in E. coli.Its broad
substrate range includes aliphatic and aromatic aldehydes as
well as ketones.[14] As the first R-specific HNL based on an
a/b-hydrolase fold, its structure will provide valuable infor-
mation concerning the enzyme mechanism of a/b-hydrolase
fold based HNLs.
Enantioselective Synthesis, Wiley-VCH, Weinheim, 1997.
[2] a) A.Glieder, R.Weis, W.Skranc, P.Pöchlauer, I.Dreveny, S.
Majer, M.Wubbolts, H.Schwab, K.Gruber, Angew. Chem. 2003,
115, 4963 – 4966; Angew. Chem. Int. Ed. 2003, 42, 4815; b) S.
Nanda, Y.Kato, Y.Asano, Tetrahedron 2005, 61, 10908; c) J.
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Curr.
Org. Chem. 2006, 10, 2037; d) R.Weis, R.Gaisberger, W.Skranc,
K.Gruber, A.Glieder, Angew. Chem. 2005, 117, 4778; Angew.
Chem. Int. Ed. 2005, 44, 4700; e) PaHNL is cofactor dependent
and requires posttranslational modifications, such as formation
of a disulfide bond and glycosylations.
[3] a) J.Albrecht, I.Jansen, M.R.Kula, Biotechnol. Appl. Biochem.
1993, 17, 191; b) M.Hasslacher, M.Schall, M.Hayn, H.Griengl,
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Arch. Biochem.
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Angew. Chem. 1996, 108, 493; Angew. Chem. Int. Ed. Engl. 1996,
35, 437; b) M.Hasslacher, M.Schall, M.Hayn, R.Bona, K.
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Hernandez, H.Luna, F.Huis-Teran, A.Vazquez, J. Mol. Catal. B
2004, 30, 105.
[6] B.Reiter, A.Glieder, D.Talker, H.Schwab,
Appl. Microbiol.
Biotechnol. 2000, 54, 778.
[7] U.Wꢀspi, B.Misteli, M.Hasslacher, A.Jandrositz, SD. .
Kohlwein, H.Schwab, R.Dudler, Eur. J. Biochem. 1998, 254, 32.
[8] Mandelonitrile cleavage was measured according to Bauer
et al.;[12] cleavage of other cyanohydrins was detected with an
HCN-based assay system.[13]
[9] For experimental details see the Supporting Information.
[10] L.M.van Langen, F.van Rantwijk, R.A.Sheldon, Org. Process
Res. Dev. 2003, 7, 823.
Received: April 4, 2007
Revised: May 20, 2007
Published online: October 1, 2007
[11] a) K.Gruber, G.Gartler, B.Krammer, H.Schwab, C.Kratky,
Biol. Chem. 2004, 279, 20501; b) G.Gartler, C.Kratky, K.
Gruber, J. Biotechnol. 2007, 129, 87.
J.
Keywords: cyanohydrins · enzyme catalysis ·
.
[12] M.Bauer, H.Griengl, W.Steiner, Biotechnol. Bioeng. 1999, 62,
genetic engineering · hydroxynitrile lyases · oxynitrilases
20.
[13] J.Andexer, J.K.Guterl, M.Pohl, T.Eggert,
Chem. Commun.
2006, 4201.
[1] a) M.Sharma, NN. .Sharma, TC. .Bhalla,
Enzyme Microb.
[14] AtHNL and its application have been filed for patent (J.
Andexer, T.Eggert, evocatal GmbH, German patent application
DE 102006058373.6, 2006).
Technol. 2005, 37, 279; b) H.Griengl, H.Schwab, M.Fechter,
Trends Biotechnol. 2000, 18, 252; c) M.H.Fechter, H.Griengl,
Food Technol. Biotechnol. 2004, 42, 287; d) M.North, Tetrahe-
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Angew. Chem. Int. Ed. 2007, 46, 8679 –8681
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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