Molecular cloning and characterization of copper amine oxidase from Huperzia serrata

Article abstract of DOI:10.1016/j.bmcl.2012.07.102

A cDNA encoding a novel copper amine oxidase (CAO) was cloned and sequenced from the Chinese club moss Huperzia serrata (Huperziaceae), which produces the Lycopodium alkaloid huperzine A. A 2043-bp open reading frame encoded an Mr 76,854 protein with 681 amino acids. The deduced amino acid sequence shared 44-56% identity with the known CAOs of plant origin, and contained the active site consensus sequence of Asn-Tyr-Asp/Glu. The phylogenetic tree analysis revealed that HsCAO from the primitive vascular plant H. serrata is closely related to Physcomitrella patens subsp CAO. The recombinant enzyme, heterologously expressed in Escherichia coli, catalyzed the oxidative deamination of aliphatic and aromatic amines. Among them, the enzyme accepted cadaverine as the best substrate to catalyze the oxidative deamination to Δ¹-piperideine, which is the precursor of the Lycopodium alkaloids. Furthermore, a homology modeling and site-directed mutagenesis studies predicted the active site architecture, which suggested the crucial active site residues for the observed substrate preference. This is the first report of the cloning and characterization of a CAO enzyme from the primitive Lycopodium plant.

Full text of DOI:10.1016/j.bmcl.2012.07.102

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5784–5790
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Molecular cloning and characterization of copper amine oxidase
from Huperzia serrata
Jieyin Sun a,b,c, Hiroyuki Morita a,d, , Guoshen Chen , Hiroshi Noguchi , Ikuro Abe
⇑
c
b
a,
⇑
a
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, Shizuoka 422-8526, Japan
Institute of Materia Medica, Zhejiang Academy of Medical Sciences, 182 Tianmushan Road, Hangzhou, Zhejiang 310013, China
Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
A cDNA encoding a novel copper amine oxidase (CAO) was cloned and sequenced from the Chinese club
moss Huperzia serrata (Huperziaceae), which produces the Lycopodium alkaloid huperzine A. A 2043-bp
open reading frame encoded an Mr 76,854 protein with 681 amino acids. The deduced amino acid
sequence shared 44–56% identity with the known CAOs of plant origin, and contained the active site con-
sensus sequence of Asn-Tyr-Asp/Glu. The phylogenetic tree analysis revealed that HsCAO from the prim-
itive vascular plant H. serrata is closely related to Physcomitrella patens subsp CAO. The recombinant
enzyme, heterologously expressed in Escherichia coli, catalyzed the oxidative deamination of aliphatic
Received 22 June 2012
Revised 27 July 2012
Accepted 30 July 2012
Available online 7 August 2012
Keywords:
Huperzia serrate
Huperziaceae
Molecular cloning
Lycopodium alkaloid
Copper amine oxidase
and aromatic amines. Among them, the enzyme accepted cadaverine as the best substrate to catalyze
1
the oxidative deamination to
D
-piperideine, which is the precursor of the Lycopodium alkaloids. Fur-
thermore, a homology modeling and site-directed mutagenesis studies predicted the active site architec-
ture, which suggested the crucial active site residues for the observed substrate preference. This is the
first report of the cloning and characterization of a CAO enzyme from the primitive Lycopodium plant.
Ó 2012 Published by Elsevier Ltd.
Copper amine oxidases (CAOs) (EC 1.4.3.21) are quinoenzymes,
and are widely distributed in bacteria, yeasts, fungi, plants and
poorly understood, various studies, including an EST analysis,12,13
suggested that it is initiated by the decarboxylation of lysine to form
1
–6
1
animals. The enzymes catalyze the oxidative deamination of pri-
mary amines to the corresponding aldehydes, with the concomitant
reduction of dioxygen to hydrogen peroxide via a ping-pong mech-
anism involving a covalently bound redox cofactor, 2,4,5-trihydr-
oxyphenylalanine quinone (TPQ), and a copper ion Cu2 (Fig. 1A).
The CAOs are homodimeric proteins consisting of 70–90 kDa sub-
units, each of containing a single copper ion and the covalently
bound cofactor TPQ, formed by the post-translational modification
of the CAO’s conserved tyrosine side chain within the consensus
cadaverine, with the subsequent formation of
D
-piperideine,
9
which is catalyzed by CAO (Fig. 1B). However, CAO genes involved
in the biosynthesis of the Lycopodium alkaloids have been still
uncovered. In order to shed light on the biosynthesis of the Lycopo-
dium alkaloids in H. serrata, we performed PCR screening of CAO
enzymes, by using primers based on the conserved sequences of
the known CAOs. Here we report the cloning and characterization
of a novel CAO from the primitive vascular plant H. serrata. The
+
enzyme accepted cadaverine as the best substrate to catalyze the
7
1
active site sequence of Asn-Tyr-Asp/Glu (Fig. 2). Among the mem-
oxidative deamination to
D
-piperideine, which is the precursor
bers of the Cu-TPQ class CAOs, the plant enzymes are generally cat-
of the Lycopodium alkaloids. This is the first report describing a
CAO enzyme from a primitive Lycopodium plant.
alytically more active than those from animals.8
The Lycopodium alkaloids are quinolizine, or pyridine and
The cDNA encoding a novel CAO (HsCAO) was cloned and se-
quenced from the roots of H. serrata by RT-PCR, using degenerate
primers based on the conserved sequences of the known plant
a
-pyridone type alkaloids, and a number of the alkaloids have been
9
isolated from 54 species of the Lycopodium plant. For example,
Huperzia serrata (Thunb.) Trev. (Huperziaceae, recently reclassified
by taxonomists, formerly Lycopodium serrata), produces the Lycopo-
dium alkaloid huperzine A, which is a potent inhibitor of acetylcho-
1
4
CAO enzymes. The terminal sequences of the cDNA were deter-
0
0
mined by the 3 - and 5 -RACE methods. The full-length cDNA con-
tained a 2043 bp ORF encoding an Mr 76,854 protein with 681
amino acids (Fig. 2). The nucleotide sequence has been deposited
in the GenBank database (GenBank ID: JN247732). No additional
cDNAs encoding CAO isomers were obtained in this study. The de-
duced amino acid sequence shared 44–56% identity with those of
the known plant CAOs: 56.5% identity to Solanum lycopersicum
1
0,11
linesterase and thus a promising drug for Alzheimer’s disease.
Although the biosynthesis of the Lycopodium alkaloids remains
⇑
Corresponding authors. Tel.: +81 3 5841 4740; fax: +81 3 5841 4744.
E-mail address: abei@mol.f.u-tokyo.ac.jp (I. Abe).
0
960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.07.102

J. Sun et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5784–5790
5785
Figure 1. Proposed mechanism of CAOs. (A) Overall proposed reaction mechanism from primary amine to aldehyde, catalyzed by CAOs. (B) Proposed biosynthetic pathway
1
from lysine to
D -piperideine.
Figure 2. Comparison of the primary sequences of HsCAO and other plant CAOs. The predicted secondary structures,
a-helices (rectangles), b-strands (arrows), and loops
(
bold lines) of HsCAO are also diagrammed. The secondary structure was predicted by the PredictProtein site (http://www.predictprotein.org). The CAO’s conserved
consensus sequence Asn-Tyr-Asp/Glu, and the copper binding histidine residues are marked with # and @, respectively. The Tyr, Lys, Asp, and Asn residues, which are thought
to play a crucial role in the activation of catalytic center TPQ, are marked with $. The regions that employed to design the degenerated primers F1, F2, R1 and R2 were
indicated with closed squares. Abbreviations (GenBank accession numbers): HsCAO, Huperzia serrata CAO; SLAO, Solanum lycopersicum CAO (CAI39243); PPAO, Physcomitrella
patens CAO (XP_001772536); LSAO, Lathyrus sativus CAO (CAH10210); PSAO, Pisum sativum CAO (Q43077).

5
786
J. Sun et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5784–5790
Figure 3. Phylogenetic analysis of plant and bacterial CAO enzymes. HsCAO is highlighted by an arrow. The optimal tree with the sum of branch length = 7.18335546 is
shown. Bootstrap values (%) out of 1000 resamplings are at each node. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances
used to infer the phylogenetic tree. The evolutionary distances are in the units of number of amino acid substitutions per site. The indicated scale represents 0.1 amino acid
substitutions per site.
CAO (SLAO), 54.6% identity to Physcomitrella patens CAO (PPAO),
5.4% to Lathyrus sativus CAO (LSAO), and 44.2% to Pisum sativum
protein migrated as a single band with an Mr of 78 kDa, in good
agreement with the calculated value of 77,676 Da (Fig. 4). In con-
trast, a gel-filtration experiment yielded an Mr of 162 kDa, suggest-
ing that the recombinant HsCAO is a homodimeric enzyme, as in
4
CAO (PSAO). A sequence analysis revealed that HsCAO contains
7
the conserved active site consensus sequence, Asn-Tyr-Asp/Glu.
1
,2,7
The active site tyrosine side chain within the Asn-Tyr-Asp/Glu se-
quence is the post-translationally modified to form the covalently-
bound co-factor, TPQ. In addition, HsCAO contains the three histi-
dine residues (His465, His467 and His627) for copper ion binding,
as well as the two active-site residues (Tyr308 and Asp322) con-
served in the CAO family enzymes. Furthermore, a phylogenetic
tree analysis revealed that HsCAO, from the primitive vascular
plant H. serrate, is closely related to Physcomitrella patens subsp
the case of the known CAOs.
As previously reported for the copper-containing amine oxi-
dases (phenethylamine oxidase and histamine oxidase from Arth-
1
8,19
robacter globiformis),
the catalytically active, recombinant
, and
HsCAO protein was obtained only in the presence of CuSO
4
was present in the soluble fraction when expressed in E. coli BLR.
In fact, when the catalytically active recombinant HsCAO was re-
2
0
acted with phenylhydrazine, which is the conventional method
to detect the covalently bound Cu-TPQ cofactor at the active site,
a UV spectra analysis revealed a peak with maximal absorption
at 448 nm, corresponding to the phenylhydrazone, as previously
1
5
CAO (Fig. 3).
The full-length cDNA of HsCAO was sub-cloned into the
pET22b(+) vector, and the recombinant HsCAO protein, with a
hexahistidine-tag at the C-terminus, was expressed in Escherichia
1
,2,5,7,21
observed for the known CAOs.
CuSO
4
In contrast, the absence of
, the inactive, Cu -free recombinant HsCAO protein did
not afford the peak in the phenylhydrazine reaction. These
2
+
coli BLR and BL21(DE3)pLysS, and purified to homogeneity by
Ni-chelating affinity column chromatography.1
6,17
The purified

J. Sun et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5784–5790
5787
Table 1
Steady-state kinetic parameters of recombinant HsCAO
cat (sꢀ1)
ꢀ1
ꢀ1
Substrate
K
M
(mM)
K
M
Kcat/K (s mM )
H2N
NH2
0
.3
.2
1.0
1.9
3.3
1.6
cadaverine
NH2
H N
2
1
putrescine
H
N
NH2
H N
2
0
0
.8
.7
2.3
1.8
2.9
2.6
spermidine
N
NH
H N
2
histamine
H N
2
0
.5
.9
1.4
3.1
2.8
1.6
benzylamine
N
H N
2
1
4
-aminomethylpyridine
N
Figure 4. SDS–PAGE of recombinant HsCAO purified by Ni-chelating affinity
column chromatography. The arrow indicates the recombinant HsCAO, at 78 kDa.
M, protein molecular weight markers; lane 1, protein solution after Ni-chelating
affinity column purifications.
H N
2
1.2
1.9
2.9
1.6
1.7
3-aminomethylpyridine
N
observations suggested that the catalytically active, recombinant
HsCAO is a copper/TPQ-containing homodimeric enzyme, and the
active site tyrosine side chain within the Asn-Tyr-Asp/Glu
sequence is post-translationally modified to form the covalently-
bound cofactor TPQ.
H2N
1.7
2-aminoethylpyridine
An LC–ESIMS analysis22 revealed that the catalytically active,
activity was maximal at pH 8.0, within the range of pH 6.8–8.5. In
contrast, when EDTA or the inactive form of HsCAO was added to
the enzyme reaction mixture, the piperideine-forming activity
was undetectable. Furthermore, since the CAO family enzymes
recombinant HsCAO accepts cadaverine as the amine substrate to
1
produce
D
-piperideine, which is the putative biosynthetic precur-
9
sor of the Lycopodium alkaloids (Fig. 5). The piperideine-forming
Figure 5. LC–ESIMS analysis of the enzymatic formation of D1-piperideine from cadaverine by HsCAO. Ion chromatograms extracted with m/z 84 and m/z 103 are shown.

5
788
J. Sun et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5784–5790
Figure 6. Overall structure of the homology model of HsCAO. (A) Both monomer A (blue) and B (silver) are represented by ribbon models. The catalytic center TPQ410 is
represented with an orange CPK model. The copper ion binding histidine residues His465, His467, and His627, are indicated with pink CPK models. (B and C) Comparison of
the homology model of (B) HsCAO and the crystal structure of (C) PASO. The catalytic center, TPQ410 is represented with an orange stick model. The copper ion binding
histidine residues, His465, His467, and His627, are indicated by pink stick models. The four residues, Tyr308, Lys318, Asp322, and Asn409, in HsCAO, and the equivalent four
residues in PSAO, are shown with black stick models. The copper ion molecule and the hydrogen bonds are indicated by a light-blue sphere and green dotted lines,
respectively.
are known to exhibit broad substrate specificities,1,2,23 we also
tested the aliphatic (putrescine and spermidine) and aromatic
residues in the model are in the most favored regions, and 14.4%
are in the additional allowed regions. The homology model pre-
dicted that HsCAO shares the same overall fold, consisting of a
(
histamine, benzylamine, tyramine, 4-aminomethylpyridine, 3-
aminomethylpyridine, and 2-aminoethylpyridine) primary amines
large b-sandwich domain and two
a/b domains, as in the case of
2
4
as substrates, and measured the steady-state kinetic parameters
PSAO. Upon dimerization, residues 383–386 are linked to the other
monomer to complete the wall of the active-site cavity in each
monomer. The Tyr in the catalytic center (corresponding to TPQ)
and the copper ion binding site, consisting of three histidine resi-
dues, are sterically conserved within each monomer (Tyr410 for
the catalytic center, and His465, His467 and His627 for the copper
ion binding site), and sit at the bottom of the internal active-site
cavity (Fig. 6B). In addition, Tyr, Lys, Asp and Asn, which are
thought to play crucial roles in the activation of the TPQ catalytic
center of PSAO, are present within each monomer (Tyr308,
Lys318, Asp322, and Asn409) (Fig. 2 and 7B). Therefore, HsCAO
may also catalyze the oxidative deamination of primary amines
to the corresponding aldehydes via a ping-pong mechanism, by
2
5
(
Table 1). These analyses demonstrated that HsCAO also accepts
all of the tested substrates, excepted for tyramine. Interestingly,
the steady-state kinetics analyses of HsCAO revealed
a
ꢀ
K
M
= 0.3 mM and a kcat = 1.0 s 1 for cadaverine, with respect to
the hydrogen peroxide formation activity, representing the best
catalytic efficiency (kcat/K ) among the substrates tested. This is
M
in sharp contrast to the previously reported L. sativus CAO (LSAO)
and P. sativum CAO (PSAO), as the former even accepts tyramine,
while latter exhibits better catalytic efficiency (kcat/K ) for putres-
M
cine. These observations indicated that HsCAO is functionally dif-
ferent from LSAO and PSAO, and suggested that HsCAO is
involved in the biosynthesis of the Lycopodium alkaloids. To test
this hypothesis, further metabolomic and proteomic analyses, such
as developmental or inducible variation of HsCAO in the alkaloid
biosynthesis, are required.
2
+
exploiting TPQ and Cu , as in the cases of the other CAOs. On
the other hand, the homology modeling study suggested that 14
residues lining the active-site cavity and entrance of PSAO are un-
iquely altered in these regions of HsCAO. Especially, the internal
active-site residues Phe140, Tyr168, Phe298, Phe304, Thr383, and
To clarify the structural basis for the HsCAO enzyme reaction, a
26
homology model was constructed, based on the X-ray crystal
structure of P. sativum CAO (PSAO).27 The model consists of resi-
dues 28–671 of both monomers A and B (Fig. 6A). In the Rama-
chandran plot calculated for the model, a total of 84.9% of the
0
Gly351 in PSAO are substituted with Ile160, Phe190, Tyr320,
0
Tyr326, Ser406 and Phe384 in HsCAO, respectively (Fig. 6B and
C), which may account for the different substrate specificities

J. Sun et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5784–5790
5789
0
and catalytic activities between these enzymes, as shown in the
steady-state kinetics studies.
TCCGTCTGAAA CTCCCA-3 ), to amplify an approximately 1.6 kb DNA fragment.
The nucleotide sequence was determined by using an ABI Prism 310 Genetic
Analyzer (Applied Biosystems). The active site was investigated via the PDB site
To test this hypothesis, we constructed a set of point mutants of
HsCAO (I160F, F190Y, Y320F, Y326F, S406T and F384G), and inves-
(http://www.pdb.org/).
15. Amino acid sequences were aligned using ClustalW (Thompson, J. D.; Higgins,
D. G.; Gibson, T. J. Nucleic Acids Res. 1994, 22, 4673), and phylogenetic tree was
constructed using MEGA5 (Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.;
Nei, M.; Kumar, S. Methods. Mol. Biol. Evol. 2011, 28, 27319) with UPGMA
method. Evolutionary distances were computed with the Poisson correction
method. In total, 37 amino acid sequences of HsCAO and other amine oxidases
from different species were assessed. All positions containing gaps and missing
data were eliminated. There were a total of 467 positions in the final dataset.
Bootstrap tests with 1000 replications were conducted to examine the
reliability of the interior branches and the validity of the trees obtained.
2
8
tigated the mechanistic consequences of the mutagenesis. The
mutants were thus expressed in E. coli, at levels comparable to that
of the wild type enzyme, and purified to homogeneity by a Ni-che-
late affinity column. The enzymatic activities of the mutants were
evaluated with the hydrogen peroxide assay by using the sub-
strates used for wild type HsCAO. All of the mutants lost the activ-
ity within the range of pH 6.8–8.5, suggesting that the residues are
crucial for the catalytic activities of HsCAO. Presumably, the single
substitution of the residues resulted in conformational changes in
the active site, which lead to the loss of the enzyme activity.
This is the first report of the cloning and characterization of a
novel CAO enzyme from the primitive club moss H. serrata, which
produces the Lycopodium alkaloid huperzine A, a potent inhibitor
of acetylcholinesterase. The deduced amino acid sequence shares
1
6. The full-length cDNA encoding HsCAO was amplified using the 1st strand
0
cDNA as the template, with a forward (5 -ATTACATATGAGCCAATCCCCAGACG
0
0
TAATAG-3
) primer (the Nde I site is underlined) and a reverse (5
-ATAAC
0
TCGAGCCTCTCGTCAACATGGCTGC-3 ) primer (the Xho I site is underlined). The
amplified DNA fragment was digested with Nde I/Xho I and cloned into the Nde
I/Xho I sites of the pET22b(+) expression vector (Novagen), for expression as a
fusion protein with a hexahistidine-tag at the C-terminus. After confirmation
of the sequence, the resultant expression plasmid was transformed into E. coli
BLR and BL21(DE3)pLysS. The cells harboring the plasmid were cultured to an
OD600 of 0.6 in LB medium containing 100
Isopropylthio-b- -galactopyranoside (IPTG; final, 1.0 mM) and CuSO
50 M) were then added to the culture medium, to induce gene expression and
l
g/ml of ampicillin at 30 °C.
4
4–56% identity with the known plant CAOs, and contains the ac-
D
4
(final,
tive site consensus sequence of Asn-Tyr-Asp/Glu. Furthermore,
functional analyses demonstrated that HsCAO exhibits the best
substrate specificity for cadaverine, which is the proposed biosyn-
thetic precursor of the Lycopodium alkaloids. Although further
metabolomic and proteomic studies are needed, this report con-
tributes to the clarification of the pooly understood biosynthetic
machinery of the Lycopodium alkaloids.
l
to activate the expressed recombinant HsCAO, respectively, and the culture
was incubated for a further for 16 h at 16 °C.
1
7. All of the following procedures were performed at 4 °C. The E. coli cells were
harvested by centrifugation at 5000 g and resuspended in 50 mM Tris–HCl pH
.0. The cells were disrupted by sonication, and the lysate was centrifuged at
2,000 g for 30 min. The supernatant thus obtained was loaded onto a Ni
Sepharose 6 Fast Flow affinity column (GE Healthcare) equilibrated with the
buffer A (40 mM potassium phosphate (KPB), pH 7.9, containing 0.1 M NaCl and
mM imidazole). After washing the column with buffer B1 (20 mM KPB, pH 7.9,
8
1
5
containing 0.5 M NaCl) and buffer B2 (20 mM KPB, pH 7.9, containing 0.2 M NaCl
and 40 mM imidazole), the recombinant HsCAO protein was eluted with 15 mM
KPB buffer pH 7.5, containing 0.5 M imidazole and 10% glycerol. Finally, the
protein solution was dialyzed against 50 mM Tris–HCl buffer pH 8.0, containing
Acknowledgments
This work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science
and Technology, Japan. The authors declare that they have no com-
peting financial interests.
10% glycerol. The protein concentration was determined by the Bradford method
(
Protein Assay Dc, Bio-Rad) using bovine gamma globulin as the standard.
18. Tanizawa, K.; Matsuzaki, R.; Shimizu, E.; Yorifuji, T.; Fukui, T. Biochem. Biophys.
Res. Commun. 1994, 199, 1096.
9. Choi, Y. H.; Matsuzaki, R.; Fukui, T.; Shimizu, E.; Yorifuji, T.; Sato, H.; Ozaki, Y.;
1
Tanizawa, K. J. Biol. Chem. 1995, 270, 4712.
References and notes
2
0. The phenylhydrazine assay was performed, according to the described
4
method. The purified recombinant HsCAO (50
l
M) was dissolved in 1 ml of
1
2
.
.
Klinman, J. P.; Mu, D. Annu. Rev. Biochem. 1994, 63, 299.
Wertz, D. L.; Klinman, J. P. In Handbook of Metalloproteins; Messerschmidt, A.,
Huber, R., Poulos, T., Wiegahardt, K., Eds.; John Wiley: New York, 2001; Vol. 2, p
0.1 M potassium phosphate buffer, pH 7.2, and then two molar equivalents of
phenylhydrazine hydrochloride (Wako Pure Chemical Industries) were added
to the enzyme solution. The mixture was incubated at 37 °C for 30 min, and
phenylhydrazone formation was then monitored in the range of 300–700 nm,
by using a UV-1700 UV–VIS Spectrophotometer (SHIMADZU).
1
258.
3
.
Parsons, M. R.; Convery, M. A.; Wilmot, C. M.; Yadav, K. D. S.; Blakeley, V.;
Corner, A. S.; Phillips, S. E. V.; McPherson, M. J.; Knowles, P. F. Structure 1995, 3,
21. Chang, C. M.; Klema, V. J.; Johnson, B. J.; Mure, M.; Klinman, J. P.; Wilmot, C. M.
1
171.
Biochemistry 2010, 49, 2540.
4
5
.
.
Cai, D.; Klinman, J. P. Biochemistry 1994, 33, 7647.
Koyanagi, T.; Matsumura, K.; Kuroda, S.; Tanizawa, K. Biosci. Biotechnol.
Biochem. 2000, 64, 717.
Rossi, A.; Petruzzeli, R.; Agro, A. F. FEBS Lett. 1992, 301, 253.
Brazeau, B. J.; Johnson, B. J.; Wilmot, C. M. Arch. Biochem. Biophys. 2004, 428, 22.
Bellelli, A.; Morpurgo, L.; Mondovì, B.; Agostinelli, E. Eur. J. Biochem. 2000, 267,
22. The reaction mixture (final volume of 50
ll) contained 100 nmol cadaverine
and 10 g of the purified recombinant HsCAO in 50 mM Tris–HCl buffer, pH
l
8.0. Incubations were performed at 25 °C for 16 h. The products were then
extracted with methanol, and separated by reverse-phase HPLC on a Shiseido
ODS C8 column (4.6 ꢁ 150 mm), at a flow rate of 0.2 ml/min. Gradient elution
6.
7.
8.
2
was performed with H O and acetonitrile, both containing 1% acetic acid: 0-
3
264.
20 min, linear gradient from 40 to 70% acetonitrile; 20–40 min, 70%
acetonitrile; 40-60 min, linear gradient from 70 to 100% acetonitrile. Online
LC–ESIMS spectra were measured with an Agilent Technologies HPLC 1100
9
1
1
.
Ma, X.; Gang, D. R. Nat. Prod. Rep. 2004, 21, 752.
0. Desilets, A. R.; Gickas, J. J.; Dunican, K. C. Ann. Pharmacother. 2009, 43, 514.
1. Luo, H.; Sun, C.; Li, Y.; Wu, Q.; Song, J.; Wang, D.; Jia, X.; Li, R.; Chen, S. Physiol.
Plant. 2010, 139, 1.
2. Ayer, W. A. Nat. Prod. Rep. 1991, 8, 455.
3. Hemscheidt, T. Top. Curr. Chem. 2000, 209, 175.
4. The H. serrata plant used in this study was obtained from Guoshen Chen (Zhejiang
Academy of Medical Science, Hangzhou, China). Total RNA was extracted from
roots of H. serrata by using an RNeasy plant mini kit (Qiagen), and was reverse-
transcribed by using the SuperScript First-Strand Synthesis System Kit for RT-PCR
series HPLC coupled to
a Bruker Daltonics Esquire4000 ion trap mass
spectrometer fitted with an ESI source. The ESI capillary temperature and the
capillary voltage were 320 °C and 4.0 V, respectively. The tube lens offset was
set to 20.0 V. All spectra were obtained in the positive mode, over a mass range
of m/z 50–600, and at a rate of one scan every 0.2 s. The collision gas was
helium, and the relative collision energy scale was set at 30.0% (1.5 eV).
23. Pietrangeli, P.; Federico, R.; Mondovi, B.; Morpuro, L. J. Inorg. Biochem. 2007,
101, 997.
1
1
1
(
Invitrogen). The single strand cDNA thus obtained was used as the template for
24. Tyramine, spermidine trihydrochloride, benzylamine and histamine dihydro
chloride were purchased from Nacalai Tesque, Inc. Cadaverine dihydrochloride
and putrescine were purchased from Sigma. 2-Aminoethylpyridine, 4-ami
nomethylpyridine and 3-aminomethylpyridine were purchased from Wako
Pure Chemical Industries.
25. Steady-state kinetic parameters were determined by using Hydrogen Peroxide
Assay kit (Biovision Incorporated). The experiments were performed using six
concentrations of substrate (0.1, 0.2, 0.5, 1, 2, 4 mM) in the assay mixture,
the PCR reactions with degenerate oligonucleotide primers, which were designed
based on the conserved sequences of the known plant copper amine oxidase, as
follows: AOF1 = 5 -TGGGC(A/C/T)(A/G)A(C/T)TGGAA(A/G)TT(C/T)C-3 , AOF2 = 5 -
AC(T/C/A/G)GT(T/C/A/G)GG(T/C/A/G)AA (T/C)TA(T/C)GA(T/C/G)T-3 , AOR1 = 5 -
GGCAT(T/C/A)A(T/C)(T/C/A/G)GG(C/A/G)(C/A)A(T/G)TC(T/C)TC-3 , AOR2 = 5 -(T/
C)GT(A/G)CCACA(T/C/A/G)(C/A)AC(T/A/G)AT(A/G)TC-3 . Nested PCR was per
formed with the AOF1 and AOR1 primer sets, and then with AOF2 and AOR2, to
amplify the core fragment. The 3 -RACE, using two gene-specific primers (SRT-
AOF1: 5 -TGGGAGTTTCAGACGGATGGCA-3 , SRT-AOF2: 5 -ACAAGAGTGAACGAT
0
0
0
0
0
0
0
0
0
containing 10 lg of the purified enzyme, in a final volume of 50 ll of 50 mM
Tris–HCl pH 8.0. The reactions were incubated at 25 °C for 10 min. The
reactions with boiled enzyme were used as the negative control. The Reaction
0
0
0
0
0
GGGCTGGTG-3 ) and anoligo-dTprimer(race32:5 -GGCCACGCGTCGACTAGTACT
TTTTTTTTTTTTTTTT-3 ) was used to amplify a 642 bp DNA fragment. The 5 -RACE
was performed using the 5´ RACE System for Rapid Amplification of cDNA Ends
0
0
Mix Solution (50
concentration was measured at OD570
protocol. Lineweaver-Burk plots of data were employed to derive the apparent
M
K and kcat values, using the Microsoft Excel program (Microsoft).
l
l/test) was then added into the reaction mixture, and the
2
H O
2
,
according to manufacturer’s
0
(
Invitrogen)andthree specific primers, SRT-AOR1(5 -ACCCTACAGGATTCCCGAGC
0
0
0
0
C-3 ), SRT-AOR2 (5 -ACAAGCCAGTCATTCCACATTCA-3 ) and SRT-AOR3 (5 -TGCCA

5
2
790
J. Sun et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5784–5790
0 0
CATTCTCTCCAGGATGGTTTG-3 and 5 -CAAACCATCCTGGAGAGAATGGCAAGCAG
6. The model of HsCAO was generated by the SWISS-MODEL package (http://
expasy.ch/spdpv/), based on the crystal structure of PSAO (PDB ID: 1KSI). The
model quality was assessed with PROCHECK (Laskowski, R.A.; MacArthur,
M.W.; Moss, D.S.; Thornton, J.M. J. Appl. Crystallogr. 1993, 26, 283). All crystallo
graphic figures were prepared with PyMOL (DeLano Scientific, http://
www.pymol.org).
0
0
0
ACGACAT-3 ), F190Y (5 -TGGATCTGCGAATGTTTACATGAGGCCACTGGAGG-3 and
0
0
0
5 -CCTCCAGTGGCCTCATGTAAACATTCGCAGATCCA-3 ), Y320F (5 -GGATGGTATT
0
0
TCAAAACATTCATGGATTCTGGAGAG-3 and 5 -CTCTCCAGAATCCATGAATGTTTTG
0
0
AAATACCATCC-3 ), Y326F (5 -CATGGATTCTGGAGAGTTTGGATTAGGAATGCTGG-
0
0
0
0
3 and 5 -CCAGCATTCCTAATCCAAACTCTCCAGAATCCATG-3 ), F384G (5 -GGAGAC
0
0
2
7. Kumar, V.; Dooley, D. M.; Freeman, H. C.; Guss, J. M.; Harvey, I.; McGuirl, M. A.;
Wilce, M. C.; Zubak, V. M. Structure 1996, 15, 943.
ACTCAGAGGCCGGTGTCCAAGATTTTGAG-3 and 5 -CTCAAAATCTTGGACACCGGC
0
0
CTCTGAGTGTCTCC-3 ), and S406T (5 -TGGTCCGAATGGTTGGAACAGTGGGGAATT
0
0
0
2
8. The plasmids expressing the HsCAO mutants (I160F, F190Y, Y320F, Y326F, F384G,
and S406T) were constructed with a QuikChange Site-Directed Mutagenesis Kit
ATGAC-3 and 5 -GTCATAATTCCCCACTGTTCCAACCATTCGGACCA-3 ). The mutant
enzymes were expressed and purified with the same procedures as described for
the wild type, and used for the Hydrogen Peroxide Assay.
(
Stratagene), according to the manufacturer’s protocol, using the following pairs
0
of primers (mutated codons are underlined): I160F (5 -ATGTCGTCTGCTTGC