Stereochemistry of 2-phenylethylamine oxidation catalyzed by bacterial copper amine oxidase

Article abstract of DOI:10.1271/bbb.67.2664

The stereochemical course of the reaction catalyzed by a copper amine oxidase from Arthrobacter globiformis has been investigated using 2-phenylethylamine stereospecifically deuterium-labeled at the C1 position. Measurements of deuterium content in the product, phenylacetaldehyde, by gas chromatography-mass spectrometry revealed stereospecific abstraction of the pro-S hydrogen during the enzymatic oxidation, as predicted from the structure modeling for the enzyme-bound substrate.

Full text of DOI:10.1271/bbb.67.2664

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Stereochemistry of 2-Phenylethylamine Oxidation
Catalyzed by Bacterial Copper Amine Oxidase
a
a
a
b
Mayumi UCHIDA , Akifumi OHTANI , Naoki KOHYAMA , Toshihide OKAJIMA , Katsuyuki
b
a
TANIZAWA & Yukio YAMAMOTO
a
Graduate School of Human and Environmental Studies, Kyoto UniversityKyoto, Kyoto
06-8501, Japan
6
b
Institute of Scientific and Industrial Research, Osaka UniversityIbaraki, Osaka 567-0047,
Japan
Published online: 22 May 2014.
To cite this article: Mayumi UCHIDA, Akifumi OHTANI, Naoki KOHYAMA, Toshihide OKAJIMA, Katsuyuki TANIZAWA & Yukio
YAMAMOTO (2003) Stereochemistry of 2-Phenylethylamine Oxidation Catalyzed by Bacterial Copper Amine Oxidase,
Bioscience, Biotechnology, and Biochemistry, 67:12, 2664-2667, DOI: 10.1271/bbb.67.2664
To link to this article: http://dx.doi.org/10.1271/bbb.67.2664
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Biosci. Biotechnol. Biochem., 67 (12), 2664–2667, 2003
Note
Stereochemistry of 2­Phenylethylamine Oxidation Catalyzed by Bacterial
Copper Amine Oxidase
1
1
1
2
Mayumi UCHIDA, Akifumi OHTANI, Naoki KOHYAMA, Toshihide OKAJIMA,
Katsuyuki TANIZAWA, and Yukio YAMAMOTO1,õ
2
1Graduate School of Human and Environmental Studies, Kyoto University, Kyoto, Kyoto 606­8501, Japan
2Institute of Scientiˆc and Industrial Research, Osaka University, Ibaraki, Osaka 567­0047, Japan
Received July28, 2003; Accepted September 10, 2003
The stereochemical course of the reaction catalyzed
by a copper amine oxidase from Arthrobacter globifor­
mis has been investigated using 2­phenylethylamine
stereospeciˆcally deuterium­labeled at the C1 position.
Measurements of deuterium content in the product,
phenylacetaldehyde, by gas chromatography­mass spec­
trometry revealed stereospeciˆc abstraction of the pro­S
hydrogen during the enzymatic oxidation, as predicted
from the structure modeling for the enzyme­bound
substrate.
the C1 position of the substrate has been studied for
enzymes from bacteria, plants, and animals.
1
,4)
Interestingly, the speciˆcity is variable depending on
the enzyme sources and substrate amines used. For
example, the pro­S proton of dopamine is abstracted
in the reaction of pea seedling enzyme, while the
pro­R proton of the same amine is abstracted in the
4
)
reaction of porcine plasma enzyme. On the other
hand, the bovine plasma enzyme is non­stereospeciˆc
for dopamine but is pro­S speciˆc for another sub­
4)
strate, benzylamine. In view of the highlyconserved
5
,6)
Key words: 2­phenylethylamine; copper­containing
amine oxidase; topa quinone; stereo­
speciˆty; deuterium­labeled substrate
active­site structures of this enzyme family,
the
origin of the inconsistent stereospeciˆcityremains to
be settled. Toward unequivocal elucidation of the
molecular basis for the stereospeciˆc proton abstrac­
tion, we have investigated the stereochemical course
of the reaction catalyzed by the enzyme from
Arthrobacter globiformis (AGAO), for which the
X­raycr sy tallographic structure had alreadybeen
Copper amine oxidase (EC 1.4.3.6) catalyzes
oxidation of various primaryamines, producing the
corresponding aldehydes, hydrogen peroxide, and
1
)
ammonia. An organic redox cofactor, topa quinone
TPQ), contained in the active site of the enzyme is
6
)
(
solved. We report here that the pro­S proton of
2­phenylethylamine is stereospeciˆcally abstracted
during the AGAO reaction and this stereospeciˆcity
is consistent with the structure model for the
substrate SchiŠ­base intermediate.
post­translationallygenerated byself­processed
modiˆcation of a speciˆc tyrosine residue in the
2
)
presence of cupric ion and molecular oxygen. The
cupric ion is essential for both TPQ generation and
cataly sis. The cataly tic cy cle of the enzy me is divided
into the reductive and oxidative half­reactions
To structurallypredict the stereospeciˆcityof
proton abstraction, 2­phenylethylamine bound to the
C5 carbonyl group of TPQ in the substrate SchiŠ­
base form was modeled in the AGAO crystal struc­
ture (PDB entry1IU7) using a commercial software
package Insight II, version 98 (Accelrys Inc., San
Diego, CA). After the two prochiral hydrogen atoms
at the C1 position of 2­phenylethylamine were gener­
ated using the Builder module, energyminimization
was done with the Discoverä3 module for the zone
within 3 Å from TPQ under a constant valance force
ˆeld. In the ˆnal model thus obtained (Fig. 1), several
residues around the phenyl ring of substrate (e.g.,
Tyr302, Phe407, and Phe105) slightly changed their
conformations to accommodate the phenyl ring in
3
)
depending on the redox state of TPQ. In the former
reductive half­reaction, the C5 carbonyl group of
TPQ reacts with the amino group of the substrate to
form the substrate SchiŠ­base, which is then convert­
ed to the product SchiŠ­base through stereospeciˆc
proton abstraction from the substrate C1 position by
an invariant aspartic acid residue (Scheme 1, A). In
the latter oxidative half­reaction, the reduced TPQ
is re­oxidized bydiox yg en, during which the cupric
|
|
ion serves as a binding site for 1e and 2e ­reduced
dioxygen species to be e‹ciently protonated and
3
)
released.
The stereospeciˆcityof proton abstraction from
õ
To whom correspondence should be addressed. Fax: {81­75­753­2999; E­mail: yukio—ˆscher.jinkan.kyoto­u.ac.jp
Abbreviations: TPQ, topa quinone; GC­MS, gas chromatography­mass spectrometry; t
mis amine oxidase
R
, retention time; AGAO, Arthrobacter globifor­

Stereochemistryof Copper Amine Oxidase
2665
the hydrophobic pocket; after the modeling other
residues did not move from the initial positions.
Most importantly, the distance between one of the
oxygen atoms of the side chain carboxyl group of
the putative catalytic base, Asp298, and the two
prochiral hydrogen atoms is shorter for the pro­S
hydrogen (3.56 Å) than for the pro­R hydrogen
phenylethylamine were prepared from L­phenylala­
nine by using tyrosine decarboxylase from Strep­
tococcus faecalis, which catalyzes decarboxylation of
aromatic amino acids with retention of conˆgura­
6
)
7)
tion, as shown in Scheme 1, B. To obtain (R)­[1­
2
H]­2­phenylethylamine, sodium phosphate buŠer
(400 mM, pH 5.5, 5 ml) was evaporated and the
residue and L­phenylalanine (330 mg) were dissolved
(
5.20 Å). In addition, the pro­S hydrogen is oriented
in a more advantageous direction to be abstracted by
Asp298 than the pro­R hydrogen. In this way, the
structure modeling suggests the pro­S speciˆc proton
abstraction.
in D
2
O (5 ml), followed byevaporation of the
solution again. After dissolving the residue in D
2
O
(10 ml), tyrosine decarboxylase (12.5 units) and
pyridoxal 5?­phosphate (1 mg) were added to the
solution and the mixture was incubated at 379C for
Two enantiomers of mono­deuterated 2­
3
1
days. The reaction mixture was neutralized with
5z NaOH and product amine was extracted with
2
CHCl
3
to give (R)­[1­ H]­2­phenylethylamine
1
(
(
(
29 mg, 12z); H NMR (270 MHz, CDCl ) d: 1.3
s, 2H), 2.74 (d, J6.6, 2H), 2.95 (m, 1H), 7.2–7.3
m, 5H). The content of the mono­deuterated amine
3
was À97z bygas chromatograph­mass spectro­
metry(GC­MS) [retention time ( t ), 10.7 min].
R
2
For synthesizing (S)­[1­ H]­2­phenylethylamine,
a mixture of L­phenylalanine (1.3 g), pyridoxal
hydrochloride (0.16 g), and D
16 mmol) was re‰uxed for 2 h. After the mixture
was freeze­dried, the residue was dissolved in
O (10 ml) and re‰uxed again for 2 h. When the
2
O (10 ml)­NaOD
(
D
2
mixture was neutralized with 3 N HCl (pH¿5)
2
with ice­cooling, DL­[2­ H]­phenylalanine precipitat­
ed as crystals. The crystals were collected by ˆltration
and washed with cold water and methanol. The
product was dissolved in 3 N HCl and treated with a
small amount of activated charcoal. After the ˆltrate
was neutralized with 15z NaOH, addition of
2
methanol with ice­cooling aŠorded DL­[2­ H]­
1
phenylalanine (0.63 g, 47z); H NMR (270 MHz,
D
2
O) d: 2.82–2.98 (AB­system, J13.5, 2H),
7
.3–7.4 (m, 5H). The non­labeled phenylalanine was
1
not detected by H NMR. Decarboxylation of DL­[2­
Scheme 1.
2
H]­phenylalanine (0.33 g, 2 mmol) in H O by the
2
Fig. 1. Stereo Diagram of Structure Model for Substrate SchiŠ­base Complex.
Active­site residues of AGAO and 2­phenylethylamine bound to the C5 carbonyl group of TPQ with the two prochiral hydrogen
atoms at the C1 position of substrate in the ˆnal model obtained byenergyminimization are shown in ball and stick models. The
distances between the two prochiral hydrogen atoms and one of the carboxyl oxygen atoms of Asp298 (catalytic base) are also shown in
9)
Å. This diagram was prepared using MolScript.

2666
M. UCHIDA et al.
Table 1. Oxidation of Deuterium­labeled Chiral Amines by AGAO
Relative peak intensity at mWz
Conˆguration of
b)
substrate Wp roduct
Major component (z)
starting amine
1
19120
121
122
123
0.098
—
1.000
0.088
1.000
—
a)
Non­labeled
amine
aldehyde
amine
aldehyde
amine
aldehyde
—
0.021
—
—
—
0.118
1.000
—
—
0.125
—
0.110
—
a)
1.000
—
0.095
0.101
1.000
0.085
0.087
2
a)
(
(
R)­[1­ H]
R­CHDNH
R­CDO (À99)
R­CHDNH
(À99)
R­CHO (À99)
2
(À97)
a)
º0.001
2
a)
S)­[1­ H]
—
1.000
2
a)
0.033
a)
{
Molecular ion peak, M
.
b)
Percent contents of the major component shown in parentheses were calculated from the mass number of the molecular ion peak and the peak intensity,
which was rectiˆed with the concomitant M|1 and M{1 peaks. R, C
CH
6
H
5
2
.
same enzymatic procedure as described above aŠord­
the labeled amine (content, À99z). Because this
process suŠers from a primary isotope eŠect, a
considerable amount of the labeled aldehyde should
be detected if the stereospeciˆcity is incomplete.
Thus, it is concluded that the AGAO oxidation of 2­
phenylethylamine proceeds in a highly stereospeciˆc
manner, exclusively abstracting the pro­S hydrogen
at the C1 position of substrate amine, as predicted
from the modeling. The modeling also suggests that
the stereospeciˆcity could change by the binding
mode of the distal part of amine substrate in the
hydrophobic pocket, composed of residues that are
2
1
ed (S)­[1­ H]­2­phenylethylamine (30 mg, 13z); H
NMR (270 MHz, CDCl ) d: 1.3 (s, 2H), 2.74 (d, J
.6, 2H), 2.95 (m, 1H), 7.2–7.3 (m, 5H). The content
of the mono­deuterated amine was À99z by GC­
MS (t 10.7 min).
3
6
R
GC­MS analyses were done with a Shimadzu GC­
7A equipped with DB­5MS (J & W Scientiˆc, Inc.)
1
and Shimadzu GCMS­QP5050A (starting column
temperature, 509C, maintained for 5 min; increasing
rate, 109C Wm in). The enantiomeric purities of both
the labeled amines were also assessed by converting
them to the diastereomeric amides of (|)­camphanic
5
,6)
not conserved among copper amine oxidases;
8
)
1
acid, according to Parker. In H NMR, the a­proton
rotations of the phenyl ring of substrate around the
C1–C2 bond would bring the pro­R hydrogen closer
to Asp298. To examine this hypothesis, further
studies of the stereospeciˆcity of AGAO with other
substrates are under way.
2
of the (R)­[1­ H]­amide appeared at 3.29–3.34
(
1.00H) and 3.10–3.14 (º0.006H), from which the
2
purity of the (R)­[1­ H]­amine was calculated to be
2
À99z. Similarly, the (S)­[1­ H]­amine was found to
have a purity of À92z after converting to the (S)­[1­
2
H]­amide, showing d values of 3.10–3.14 (1.00H)
References
and 3.29–3.34 (º0.08H). Before the stereochemical
analysis, both of the substrate amines were converted
to the corresponding hydrogen sulfates by treating
with sulfuric acid in methanol.
The enzyme reaction was done in 200 mM HEPES
buŠer (pH 6.8) containing 0.5 mM substrate amine
and 0.6 mg Wm l AGAO in a total volume of 1 ml. The
reaction mixture was incubated for 60 min at room
temperature and the product phenylacetaldehyde was
extracted with 0.5 ml ethyl acetate. A sample of the
1
2
)
)
McIntire, W. S., and Hartmann, C., Copper­contain­
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R

9.6 min) and the contents of the deuterium­labeled
amines and aldehydes were rectiˆed with the relative
intensities of the concomitant M|1 and M{1 peaks
observed for the non­labeled amines and aldehydes.
4
)
Coleman, A. A., Hindsgaul, O., and Palcic, M. M.,
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2
[
1­ H]­aldehyde with a deuterium content of À99z
2
from (R)­[1­ H]­amine with a deuterium content of
À97z. This result clearly indicates that the pro­S
5
)
hydrogen has been abstracted during the reaction.
This stereochemical course is also supported by the
2
experiment with (S)­[1­ H]­amine where the non­
labeled aldehyde (content, À99z) was yielded from
6) Wilce, M. C., Dooley, D. M., Freeman, H. C., Guss,

Stereochemistry of Copper Amine Oxidase
2667
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)
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