L-tyrosine β-naphthylamide is a potent competitive inhibitor of tyramine n-(hydroxycinnamoyl)transferase in vitro

Article abstract of DOI:10.1016/S0031-9422(00)00427-1

L-Tyrosine β-naphthylamide, a synthetic substrate designed to measure tyrosine aminopeptidase activity, is a potent inhibitor of hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT) purified from elicited tobacco cell-suspension cultures. T

Full text of DOI:10.1016/S0031-9422(00)00427-1

Phytochemistry 56 (2001) 523±527
www.elsevier.com/locate/phytochem
l-Tyrosine b-naphthylamide is a potent competitive inhibitor of
tyramine N-(hydroxycinnamoyl)transferase in vitro
Jonathan Negrel *, Francine Javelle
Â
UMR INRA/Universite de Bourgogne BBCE-IMP, INRA BP 86510 21065 Dijon Cedex, France
Received 19May 2000; received in revised form 12 October 2000
Abstract
l-Tyrosine b-naphthylamide, a synthetic substrate designed to measure tyrosine aminopeptidase activity, is a potent inhibitor of
hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT) puri®ed from elicited tobacco cell-suspension cultures.
The inhibition is competitive, with the inhibitor binding reversibly to the tyramine binding site of the enzyme. Similar results were
obtained with THT extracted from elicited potato cell-suspension cultures. Ki values were found to be 0.66 mM for the enzyme from
tobacco and 0.3 mM for the enzyme from potato. l-Tyrosine 7-amido-4-methylcoumarin, a ¯uorogenic substrate for tyrosine ami-
nopeptidases, the structure of which is close to that of l-tyrosine b-naphthylamide, was also a powerful inhibitor, but slightly less
e€ective with Ki values of 0.72 and 0.42 mM for tobacco and potato THT, respectively. l-Tyrosine b-naphthylamide was rapidly
hydrolysed when fed in vivo to tobacco or potato cell cultures or when incubated in crude enzymic extracts prepared from these
cultures. This hydrolysis, which is presumably catalysed by aminopeptidases, precludes the use of l-tyrosine amides as inhibitors of
THT in vivo. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Nicotiana tabacum; Solanum tuberosum; Hydroxycinnamoyl transferase; Inhibition; l-Tyrosine b-naphthylamide; l-Tyrosine 7-amido-4-
methylcoumarin; Tyrosine aminotransferase
1. Introduction
1999). In a parallel approach, we have been screening
for THT inhibitors which might be used in vivo. We
previously reported that various l-tyrosine esters are
competitive inhibitors of tobacco THT, the most active
being l-tyrosine benzyl ester (Negrel and Javelle, 1997).
In this paper we report that l-tyrosine b-naphthylamide
(l-Tyr-NA, Fig. 1), a commercially available substrate
used to measure tyrosine aminopeptidase activity, is a
remarkably powerful inhibitor of both tobacco and
potato THTs. However, when fed to tobacco or potato
cell-suspension cultures, l-Tyr-NA was rapidly hydro-
lysed, most likely by aminopeptidases. This hydrolysis
precludes the use of l-Tyr-NA as a THT inhibitor in vivo.
The enzyme hydroxycinnamoyl-CoA: tyramine N-
hydroxycinnamoyl transferase, (THT, EC 1.2.2.110)
catalyses the condensation of various hydroxycinnamoyl-
CoA esters with tyramine. The amides formed by THT
have repeatedly been found associated with cell wall
fractions and their deposition together with other phe-
nolics in the cell wall is believed to create a barrier
against pathogens by reducing cell wall digestibility
(Negrel et al., 1996; Facchini et al., 1999; McLusky et
al., 1999). THT is a rational target to attempt to inhibit
the synthesis of hydroxycinnamoyltyramines to evaluate
the role played by these amides in the reinforcement of
cell walls. THT has so far been puri®ed from potato,
tobacco, and opium poppy cell-suspension cultures
(Hohlfeld et al., 1996; Negrel and Javelle, 1997; Yu and
Facchini, 1999). Both potato and tobacco THT cDNA
clones have been isolated, creating the opportunity to
generate THT-downregulated plants using an antisense
RNA strategy (Farmer et al., 1999; Schmidt et al.,
2. Results and discussion
2.1. Inhibition of THT in vitro
During the course of our research on the inhibition of
tobacco THT by l-tyrosine derivatives, we found that
l-Tyr-NA, a synthetic substrate for tyrosine amino-
peptidase, was a very potent inhibitor of the transferase
* Corresponding author. Tel.: +33-380-633000; fax: +33-388-693263.
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00427-1

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J. Negrel, F. Javelle / Phytochemistry 56 (2001) 523±527
(Table 1). THT catalyses the transfer of ferulic acid from
feruloyl-CoA to tyramine in an ordered bi bi mechanism
that initially involves binding of feruloyl-CoA to the
enzyme (Hohlfeld et al., 1995; Negrel and Javelle, 1997).
The inhibition pattern observed in the presence of l-Tyr-
NA was similar to that previously described with l-ty-
rosine benzyl ester (Negrel and Javelle, 1997). The inhi-
bition was completely reversed in the presence of high
concentrations of tyramine (Fig. 2A), whereas uncom-
petitive inhibition was observed when increasing con-
centrations of feruloyl-CoA were added in the presence
of ®xed concentrations of tyramine (Fig. 2B). These
results show that l-Tyr-NA does not inhibit the initial
binding of feruloyl-CoA to the transferase, but instead
competes with tyramine for the same binding site. This
was further con®rmed by analysing the structure±acti-
vity relationships of several amino acid naphthylamides.
l-Tyr-NA was much more active than phenylalanine b-
naphthylamide, whereas the arginine derivative was
totally inactive (Table 1). As expected, l-Tyr-NA was
also a powerful inhibitor of the recombinant tobacco
transferase, in displaying 92% inhibition at 50 mM in
the presence of 25 mM feruloyl-CoA and 125 mM ty-
ramine. Interestingly, l-tyrosine 7-amido 4-methyl cou-
marin (l-Tyr-AMC, Fig. 1), a sensitive ¯uorogenic
substrate for tyrosine aminopeptidase (Zimmerman et
al., 1977), was also a strong inhibitor of tobacco THT,
although slightly less potent than l-Tyr-NA. Both com-
pounds inhibited potato THT, and were about 10-fold
Fig. 1. Structures of feruloyltyramine (1), l-tyrosine b-naphthylamide
(2) and l-tyrosine 7-amido-4-methylcoumarin (3).
Table 1
Inhibition of tobacco or potato THT by various amino acid amides or
esters
Ki (mM)a
Inhibitor
Tobacco
Potato
l-Tyrosine b-naphthylamide
l-Tyrosine 7-amido 4-methylcoumarin
l-Tyrosine benzyl ester
l-Phenylalanine b-naphthylamide
l-Arginine b-naphthylamide
0.66
0.72
3
0.3
0.42
3.4
91
112
b
±
±
Fig. 2. Lineweaver±Burk plots of THT activity showing the variations
of 1/V at constant and saturating concentration of one substrate (A:
[feruloyl-CoA]=25 mM; B: [tyramine]=250 mM), with varying con-
centrations of the second substrate, in the absence or presence of var-
ious concentrations of l-Tyr-NA.
a
Ki values were calculated from Dixon plots, after measuring the
inhibition of THT at di€erent inhibitor concentrations, from 10 to 200
mM.
b
No detectable inhibition.

J. Negrel, F. Javelle / Phytochemistry 56 (2001) 523±527
525
more potent than l-tyrosine benzyl ester (Table 1). The
fact that the highest inhibition was obtained with the
naphthylamine derivative is interesting because it pro-
vides a clue about the mode of action of this family of
inhibitors. The structure of l-Tyr-NA is notable
because it is intermediate between tyramine and a
feruloyltyramine analogue (Fig. 1). Taking the mechanism
of action of THT into account, it is likely that the
naphthylamide moiety of the inhibitor, which is com-
parable to a rotationally constrained analogue of ferulic
acid, can interact with the feruloyltyramine binding site
after l-Tyr-NA has bound to the tyramine binding site of
the enzyme-feruloyl-CoA complex. This likely explains
the high anity of THT for l-Tyr-NA and the fact that
l-Tyr-AMC is less e€ective than l-Tyr-NA. Thus the
mode of action of these inhibitors seems di€erent from
that of other speci®c inhibitors which have been
designed to inhibit acyl-CoA amine acyltransferases,
such as nonhydrolysable acyl-CoA analogues (Paige et
al., 1990, Rudnick et al., 1991) or multisubstrate analo-
gues (Cullis et al., 1982, Erwin et al., 1984).
Fig. 3. Time course of hydrolysis of l-Tyr-NA in a crude enzyme
extract from tobacco cells. The incubation mixture contained 1.14 mg
protein and 0.5 mmol l-Tyr-NA in a total volume of 1 ml.
2.2. Metabolism of l-Tyr-NA
We next considered the possibility of using l-Tyr-NA
as an inhibitor of THT in vivo. Since amino acid naph-
thylamides are known substrates of aminopeptidases
(Bodansky, 1971), we ®rst tried to incubate l-Tyr-NA
in crude protein extracts to detect a possible tyrosine
aminopeptidase activity. Both l-Tyr-NA and l-Tyr-
AMC were rapidly hydrolysed when incubated in
enzyme extracts from tobacco or potato cell-suspension
cultures. The hydrolysis of l-Tyr-NA was monitored
photometrically whereas hydrolysis of l-Tyr-AMC was
detected ¯uorometrically. Aliquots of the enzyme
extracts were analysed by HPLC at regular intervals to
check the nature of the products formed during the
reaction. As expected, tyrosine and b-naphthylamine
were the only products formed from l-Tyr-NA (Fig. 3)
whereas tyrosine and 7-amino-4-methylcoumarin were
rapidly formed from l-Tyr-AMC. The speci®c activities
of tyrosine aminopeptidase in crude enzyme extracts
prepared from potato or tobacco cell-suspensions one
week after sub-culture were 6 and 1 nkat/mg protein,
respectively. The rate of hydrolysis of l-Tyr-NA was
similar in extracts from elicited and non-elicited cells. l-
Tyr-NA was also hydrolysed when fed in vivo to
tobacco or potato cell-suspension cultures. When added
at a concentration of 10À4 M to the culture medium of
tobacco cell suspensions, Tyr-NA was almost com-
pletely taken up after 3 h (Fig. 4). It was then rapidly
metabolised inside the cells and became undetectable
after 8 h. At that time b-naphthylamine could be detec-
ted in cell extracts but it was also quickly metabolised
and became undetectable after 24 h (Fig. 4). Similar
results were obtained with potato cell cultures (data not
Fig. 4. Uptake and metabolism of l-Tyr-NA in tobacco cell-suspen-
sion cultures. Results are expressed as a percentage of the initial
quantity of l-Tyr-NA ( 25 mmol) added to the culture medium: l-Tyr-
NA in the culture medium (&), l-Tyr-NA (*) and b-naphthylamine
(~) extracted from the cells.
shown). Aminopeptidases are widespread enzymes in
higher plants (Taylor, 1993; Chao et al., 1999). The
rapid hydrolysis of l-Tyr-NA and l-Tyr-AMC there-
fore precludes their use in vivo as THT inhibitors.
However, it may be possible to synthesize l-Tyr-NA
analogues which would be resistant to aminopeptidases.
Potent inhibitors of choline acetyltransferase resistant
to cholinesterase have been obtained by synthesizing
keto analogues of acetylcholine (Rama Sastry et al.,
1988). Substitution of a ketone group for the amide
bond of l-Tyr-NA may prevent the hydrolysis by ami-
nopeptidases while keeping the inhibitory properties of
the molecule. The preliminary results presented in this

526
J. Negrel, F. Javelle / Phytochemistry 56 (2001) 523±527
paper show the usefulness of amino acid naphthylamides
as tools to study the mechanism of action of amine N-
hydroxycinnamoyltransferases and to identify the three-
dimensional structure of the active site of these enzymes.
channel UV detector at 280 and 328 nm. The following
elution system was applied: linear gradient elution
within 45 min from 10 to 85% MeOH in water acidi®ed
at pH 2 with 4% HCO2H and 2 g/l ammonium formiate,
using a ¯ow rate of 0.8 ml/min. Retention times: l-ty-
rosine 5.45 min, b-naphthylamine 25.65 min, 7-amino-4-
methylcoumarin 29.46 min, l-tyrosine benzylester 30.98
min, l-Tyr-AMC 32.50 min, l-Tyr-NA 36.43 min.
3. Experimental
3.1. Chemicals and substrates
3.5. Metabolism of l-Tyr-NA
l-Tyr-NA, l-Tyr-AMC, l-phenylalanine b-naphthyl-
amide, l-arginine b-naphthylamide and l-tyrosine ben-
zyl ester were all purchased from Sigma. Feruloyl-CoA
was prepared by transesteri®cation of feruloyl N-
hydroxysuccinimide ester as previously described
(Negrel and Javelle, 1997).
To monitor the degradation of l-Tyr-NA in vivo, 9.2
mg l-Tyr-NA dissolved in 2 ml MeOH 50% were added
to 250 ml tobacco or potato cell-suspension cultures 1
week after sub-culture. Samples (25 ml) were withdrawn
for analysis by pipetting from the treated cultures. Cells
were then collected by centrifugation, frozen in liquid
nitrogen and kept at À80ꢀC until analysed. Aliquots of
the supernatant were also frozen in order to monitor the
uptake of l-Tyr-NA from the culture medium. Cells
were extracted twice in MeOH acidi®ed with 1% acetic
acid (2 ml/ g fr. wt.). After centrifugation the MeOH
extracts were pooled, evaporated to dryness, and taken
up in 5 ml MeOH±H2O±AcOH (3:1:1). Aliquots (20 ml)
were then analysed by HPLC.
3.2. THT puri®cation
The inhibition of tobacco THT was studied using an
enzyme puri®ed to apparent homogeneity from cell-
suspension cultures of tobacco (Nicotiana tabacum L. cv
Xanthi), as previously described (Negrel and Javelle,
1997). The potato enzyme was partially puri®ed using
the same protocol from pronase-elicited cell-suspension
cultures of Solanum tuberosum L. (cv BF15), originally
initiated from tuber callus tissue culture. The speci®c
activity of the potato enzyme was 58 nkat/mg protein. It
was completely free of tyrosine aminopeptidase activity.
Inhibition of the recombinant tobacco enzyme was stu-
died using a crude bacterial extract obtained after lysis
of the XLOLR E. coli transformant (clone 10) in 0.1 M
Tris±HCl bu€er pH 7.5 containing 1 mg mlÀ1 lysosyme
and 10 mM ME (Negrel and Javelle, 1997).
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