(Received for publication, July 10, 1995; and in revised form, August 3, 1995)
From the
L-Tryptophan 2`,3`-oxidase, an amino acid
,
-dehydrogenase isolated from Chromobacterium violaceum, catalyzes the formation of a double bond between the C
and C
carbons of various tryptophan derivatives
provided that they possess: (i) a L-enantiomeric
configuration, (ii) an
-carbonyl group, and (iii) an unsubstituted
and unmodified indole nucleus. Kinetic parameters were evaluated for a
series of tryptophan analogues, providing information on the
contribution of each chemical group to substrate binding. The
stereochemistry of the dehydro product was determined to be a Z-configuration from proton nuclear magnetic resonance
assignments. No reaction can be observed in the presence of other
aromatic
-substituted alanyl residues which behave neither as
substrates nor as inhibitors and therefore do not compete against this
reaction. The enzymatic synthesis of
,
-dehydrotryptophanyl
peptides from 5 to 24 residues was successfully achieved without side
product formation, irrespective of the position of the tryptophan
residue in the amino acid sequence. A reactional mechanism involving a
direct
,
-dehydrogenation of the tryptophan side chain is
proposed.
Amino acid oxidases and dehydrogenases are widely distributed in
living cells where they play a key role in the metabolic fate of amino
acids, generating -keto acids by oxidative deamination or
dehydration reactions(1, 2, 3) .
In this
context, we recently isolated a novel enzyme from Chromobacterium
violaceum (ATCC 12472), and showed it to convert N-acetyl-L-tryptophanamide (NATA) ()into N-acetyl-
,
-dehydrotryptophanamide
(
NATA)(4) . We named this enzyme L-tryptophan
2`,3`-oxidase (or L-tryptophan: oxygen 2`,3`-oxidoreductase). L-Tryptophan 2`,3`-oxidase, therefore, might contribute to the
conversion of L-tryptophan into indole-3-pyruvic acid, its
-keto derivative, by catalyzing the first step of the reaction
shown in the Fig. S1. Indeed, Dakin (5) first
demonstrated in 1926 that an
,
-dehydroamino acid exists in a
tautomeric equilibrium with its imino acid form and subsequently, this
latter species was shown to be readily hydrolyzed into the
-substituted corresponding pyruvic acid (
-keto acid) (6) . Hence, as it was originally
suspected(7, 8) , the transient formation of
,
-dehydroamino acids could account for a possible mechanism
in the course of the amino acid catabolism.
Figure S1:
Scheme
1Tautomeric equilibrium of ,
-dehydrotryptophanamide leading
to spontaneous hydrolysis of the imino form in aqueous
medium.
L-Tryptophan
2`,3`-oxidase is a novel hemoprotein exhibiting a number of interesting
features(4) . First, it seems to directly catalyze the
conversion of NATA into NATA and in a stoichiometric manner the
reduction of molecular O
to generate
H
O
. Second, it possesses a high molecular
weight hetero-oligomeric structure (M
680,000) consisting of two subunits:
(M
= 14,000) and
(M
=
74,000), presumably organized in an (
)
manner
with one heme molecule per
protomer. Third, L-tryptophan 2`,3`-oxidase was shown to be active at high
temperature (up to 80 °C) and under conditions that are frequently
denaturing for proteins, i.e. 0.2 M dithiothreitol,
1% SDS, or 4.5 M urea. At present, however, little is known as
to the substrate specificity of the enzyme and to a possible reaction
mechanism.
With the view to shed light on these questions, we
investigated the capacity of L-tryptophan 2`,3`-oxidase to
react with a variety of tryptophan derivatives. Here we report
steady-state kinetic parameters determined for these reactions and
specify the stereochemistry of the dehydro product. Together, the data
obtained suggest that L-tryptophan 2`,3`-oxidase proceeds to a
direct ,
-dehydrogenation of the tryptophan side chain via a
mechanism closely related to that proposed for fatty acyl-CoA
dehydrogenases(9) , in sharp contrast with the complex
pH-dependent dichotomous mechanism postulated for the Pseudomonas tryptophan side chain oxidase (EC 1.13.99.3) (10, 11) . Finally, we demonstrate that L-tryptophan 2`,3`-oxidase can also proceed to the
,
-dehydrogenation of L-tryptophan side chains
included in a number of peptides. This finding offers new enzymatic
issues as to the modification and labeling of this peculiar side chain
in proteins.
L-Tryptophan 2`,3`-oxidase was
purified from C. violaceum (ATCC 12472) according to Genet et al.(4) , and stored at -80 °C in 0.1 M bis-Tris buffer, pH 7, in the presence of 5 mM EDTA
and 0.58 mM phenylmethylsulfonyl fluoride. Working solutions,
prepared by dilution in the same buffer, were not used for more than a
day. Using NATA as a model substrate, the molar activity of the
purified enzyme, as defined with respect to 1 mol of NATA produced
per s per mol of enzyme protomer (mole of heme), was estimated to be
equal to 44.4 ± 4.1 s
.
A number of tryptophan derivatives behaved as substrates. These are L-tryptophan, L-tryptophanamide, N-acetyl-L-tryptophan, and indole-3-propionate whose conversion exhibited an absorbance increase at characteristic wavelengths (Table 1). The intensity of these absorption bands varied from one product to another. Surprisingly, however, in the case of L-tryptophan and L-tryptophanamide, the absorbance increase was followed after 1 to 3 min of reaction by a reverse effect, i.e. an absorbance decrease at 320 or 326 nm, respectively. This phenomenon suggested the unstability of the reaction products and the inhibition of the enzyme by one of the associated side products. Such an inhibition was reversible since the enzyme was still fully active upon dilution in the presence of NATA (5 mM).
In all cases, therefore, the
steady-state kinetic parameters were accurately determined according to
classical methods. The initial velocities were evaluated during the
first 10 s of the reactions at various substrate concentrations. The
resulting conventional hyperbolic curves (Fig. 1) allowed us to
estimate the kinetic parameters by nonlinear regression analyses. Note
that in these calculations, we assumed that all products whose
unstability sometimes made their isolation difficult had the same molar
absorption coefficient as NATA (
= 18.8
mM
cm
(4) ).
Figure 1:
Determination of steady-state kinetic
parameters of various substrates for L-tryptophan
2`,3`-oxidase. Experimental conditions are given under
``Experimental Procedures.'' The total enzyme concentration, E, was 6.6 nM (mole of
heme).
The kinetic parameters of the reactions performed with the different
substrates are summarized in the Table 1. One may note that L-tryptophan 2`,3`-oxidase displays the highest efficiency for L-tryptophan, which has been proposed to be the most probable
natural substrate of the enzyme(4) . Otherwise, most substrates
did not exhibit major kinetic differences. In summary therefore,
modifications of L-tryptophan which do not abolish the
enzymatic activity of L-tryptophan 2`,3`-oxidase are blockage
of the -amino group (N-acetyl-L-tryptophan), the
-carboxylic group (L-tryptophanamide), or both (NATA), as
well as deamination (indole-3-propionate).
Figure 2:
Analysis of the reaction of L-tryptophanamide with L-tryptophan 2`,3`-oxidase.
The general experimental conditions are given under ``Experimental
Procedures.'' 1 mML-Tryptophanamide was
incubated in 50 mM succinate buffer, pH 5.6, containing 40
µg ml catalase, in the presence of 6.6 nM enzyme (mole of heme), for 15 h at 30 °C. The components of
the reaction medium were separated by reverse-phase chromatography on a
Vydac C18 column, eluted with a 30-min gradient from 0 to 40%
acetonitrile in 0.1% trifluoroacetic acid, at a flow rate of 1 ml
min
. The elution was followed at 275 (-) and
330(- - -) nm. The peaks were identified by mass spectrometry
(CI-MS).
Unambiguously, the dehydro products of N-acetyl-L-tryptophan and indole-3-propionate (Fig. 1) were, respectively, identified as N-acetyl-,
-dehydrotryptophan and indole-3-acrylate,
characterized by an absorption band at (
) 318 nm
(
= 14.8 mM
cm
) and 326 nm (
=
19.6 mM
cm
(6) ),
and by mass spectrometry (CI-MS) (m/z = 245 and 188;
[M+H]
). Mass spectrometry analyses also
revealed the presence of an additional component whose mass was
correspondingly 44 units lower. The same molecular species is also
detected in the commercially available indole-3-acrylic acid,
suggesting that these additional components were in fact generated
during the mass spectrometry experiments, resulting probably from
unwanted fragmentation (decarboxylation) associated with the mode of
ionization.
Figure S2:
Scheme 2. N-Acetyl-(Z,E)-,
-dehydrotryptophan ethyl
ester. Ac, acetyl; OEt, ethyl
ester.
Figure 3:
H NMR analysis of
enzymatically prepared N-acetyl-
,
-dehydrotryptophan
ethyl ester as compared to pure E and Z reference
compounds. The reference compounds N-acetyl-(Z,E)-
,
-dehydrotryptophan ethyl
ester were chemically synthesized according to Hengartner et
al.(14) , and then separated on reverse-phase
chromatography. The enzymatically synthesized dehydro product was
obtained by incubation of N-acetyl-L-tryptophan ethyl
ester (1 mM) with L-tryptophan 2`,3`-oxidase (6.6
nM), in 50 mM succinate buffer, pH 5.6, containing 30
µg ml
catalase, for 15 h at 30 °C.
H NMR spectra were recorded in dimethyl
sulfoxide-d
at 25 °C using a
Brücker WM250 spectrometer (250 MHz). The spectra
showed the vinyl proton region between 6.6 and 8.1 ppm. a, Z
reference compound: the chemical shift of the vinyl proton appears
at 7.60 ppm. b, E reference compound: vinyl proton at 6.90
ppm. c, enzymatically synthesized
N-acetyl-
,
-dehydrotryptophan ethylester: vinyl proton at 7.60
ppm.
To identify the stereochemistry of the dehydro
product generated by L-tryptophan 2`,3`-oxidase, we incubated
the enzyme (0.8 µg ml; heme concentration,
9.1 nm) with N-acetyl-L-tryptophan ethyl ester
(2.2 mM) and purified the resulting dehydro product on
reverse-phase chromatography under conditions described above. N-Acetyl-
,
-dehydrotryptophan ethyl ester was
characterized by an absorption band at (
) 340 nm and
by mass spectrometry (m/z = 273;
[M+H]
). As shown in Fig. 3,
H NMR spectrum of this compound showed the vinyl proton to
be shifted downfield (
= 7.60 ppm) as previously observed
in the case of the Z-isomer. No signal was found corresponding
to the E-isomeric form. Therefore, the steric configuration of
the
,
-dehydrotryptophanyl moiety produced by enzymatic
dehydrogenation was confirmed unambiguously to have a Z-geometry.
Figure 4:
Inhibition of NATA oxidation by various
tryptophan analogues. Experimental conditions are given under
``Experimental Procedures.'' NATA concentrations are shown
against the curves. The total enzyme concentration, E, was 3.6 nM (mole of
heme).
Therefore, the modifications that make a tryptophan derivative an
inhibitor of L-tryptophan 2`,3`-oxidase are: (i) alteration of
the indole ring (5-hydroxy-L-tryptophan, N-formyl-L-tryptophan), (ii)
decarboxylation (tryptamine, skatole) and inversion of the L-configuration (D-tryptophan, N-acetyl-D-tryptophan).
Figure 5:
Spectral changes associated with the
dehydrogenation of tryptophan side chain in (a) pentagastrin, (b) luteinizing hormone-releasing hormone (LH-RH), and (c) adenocorticotropic hormone (ACTH 1-24). Experiments
were performed under standard kinetic conditions at 30 °C in the
presence of catalase (30 µg ml) as described
under ``Experimental Procedures.'' Peptide and enzyme
(expressed in mole of heme) concentrations were: a, 38
µM and 11.8 nM; b, 39 µM
and 11.8 nM; c, 35 µM and 20.4
nM. Spectra were recorded after the addition of L-tryptophan 2`,3`-oxidase at regular intervals of: a, 1.3 min; b, 4 min; c, 12
min.
As
a first structural requirement for a ligand to remain a substrate is
the indole ring itself. Replacement of the indole nucleus by any other
aromatic ring makes the derivative unrecognized by L-tryptophan 2`,3`-oxidase, whereas introduction of
substituents as small as hydroxyl or formyl groups transforms the
derivative into a competitive inhibitor and causes a marked affinity
decrease for the enzyme. Presumably, therefore, L-tryptophan
2`,3`-oxidase possesses an appropriate pocket which accommodates the
indole ring of tryptophan, with at least some of constitutive atoms of
the nucleus that directly interact with, or are closely related to,
essential elements for the catalysis. A second substrate requirement
resides in the presence of the -carbonyl group. Indeed, if the
carboxylic moiety can be blocked, like in NATA, without major
consequence for the substrate, removal of the carbonyl group, like in
tryptamine and skatole, transforms the derivative into a competitive
inhibitor. Strikingly, however, shortening of the tryptophan side chain
is accompanied by a substantial increase in binding affinity of skatole
as compared to tryptamine. Presumably, therefore, the
-carbonyl
group together with the indole ring establish leading interactions for
the recognition of the ligand and/or for the expression of the
catalytic activity of L-tryptophan 2`,3`-oxidase. In sharp
contrast, the
-amino moiety can be either blocked, like in NATA,
or even removed, like in indole-3 propionate, without major consequence
for the substrate. A third substrate requirement resides in the L-enantiomeric configuration of the substrate. Upon inversion
of the L-configuration, as in D-tryptophan, the
derivative becomes a competitive inhibitor with K
approximately equal to the K
value of the
corresponding substrate.
We also unambiguously showed, on the basis
of H NMR analyses, that
,
-dehydrotryptophanyl
products that are generated by L-tryptophan 2`,3`-oxidase
adopt a pure Z-geometry (Fig. S2). This result is in
good agreement with that previously reported by Gustafson et
al.(15) , who proposed that the
carboxybenzoyl-
,
-dehydrotryptophan, which is directly
produced in growing cultures of C. violaceum, results from the syn elimination of the H
and pro-S-H
hydrogen atoms. This mechanism
appears, however, all the more striking as it is unfavorable in peptide
structure, due to steric hindrance that prevents formation of an
eclipsed conformation of the C
and C
substituents.
Two pieces of evidence suggest that L-tryptophan 2`,3`-oxidase may proceed by a direct
dehydrogenation process, involving the direct removal of two reducing
equivalents from the C and C
carbon
atoms of a tryptophan residue. First, no component other than the
,
-dehydro products themselves could ever be identified upon
reaction with L-tryptophan 2`,3`-oxidase. Second, we did not
detect any lag period before the formation of the dehydro product, in
contrast to what is observed with other enzymes such as Pseudomonas tryptophan side chain oxidase (see below) which proceeds via the
formation of a cyclic intermediate(10) . Therefore, we wish to
suggest that L-tryptophan 2`,3`-oxidase proceeds by a direct
dehydrogenation mechanism such as those that are often mediated by
flavin prosthetic groups. One should note, however, that if the
presence of a flavin has been disproved in the case of L-tryptophan 2`,3`-oxidase(4) , the nature of the
dehydrogenase cofactor and the mode of electron transfer from the
substrate to molecular oxygen remain to be elucidated.
Previous
observations now deserve to be recalled to provide a basis for a
mechanistic model that could account for L-tryptophan
2`,3`-oxidase. In 1926 Dakin et al.(5) suggested that
the initial stages of amino acid and fatty acid metabolism may be
essentially similar, and it was shown later that mitochondrial
flavoenzymes, such as acyl-CoA dehydrogenases, catalyze the
,
-dehydrogenation of fatty acyl-CoA thioesters in the initial
step of fatty acid
-oxidation. Among the acyl-CoA dehydrogenase
superfamily, the medium chain fatty acyl-CoA dehydrogenase exhibits
such a broad substrate specificity that it can tolerate aromatic group
substitution at the
position of the fatty acyl-CoA substrate. In
this context, 3-indolepropionyl-CoA was converted into
3-indoleacryloyl-CoA according to the general mechanism of all
flavin-dependent acyl-CoA dehydrogenases(16) . As shown in the Fig. S3, the reaction is initiated by abstraction of the
relatively acidic
-hydrogen as a proton, followed by the direct
transfer of the
-hydrogen as a hydride to the oxidized flavin
cofactor, yielding a trans-enoyl-CoA product(9) .
Because it is clearly compatible with its substrate structural
requirements, we suggest that L-tryptophan 2`,3`-oxidase
proceeds via a similar mechanism. One major argument in favor of this
proposal resides in the stereoselectivity of the reaction catalyzed by L-tryptophan 2`,3`-oxidase. Indeed, if the inversion of the
geometric configuration abolishes the enzymatic reaction, it does not
induce a perturbation of the binding properties. In addition, the
proposed mechanism offers a simple explanation as to the observed
critical role of the substrate carbonyl group, which, by forming a
hydrogen bond with the protein, could (i) strengthen the acidic
character, and (ii) fix the orientation of the H
of the
substrate, so as it could be easily abstracted by a chemical group of
the enzyme acting as an acid-base catalyst. Further experiments are now
required to validate our proposal.
Figure S3: Scheme 3Postulated mechanism for the dehydrogenation of 3-indole propionyl-CoA by medium chain acyl-CoA dehydrogenase. From Guisla et al.(9) and Johnson et al.(16) .
Two other enzymes are presently
known to catalyze the formation of ,
-dehydro amino acids, Pseudomonas tryptophan side chain oxidase was first reported
to catalyze the formation of
,
-dehydrotryptophan residues.
However, this enzyme is clearly distinct from L-tryptophan
2`,3`-oxidase by both its structural properties and substrate
specificity (for a review, see (11) ). Indeed, the pH-dependent
mechanism proposed for the tryptophan side chain oxidase totally
differs from that proposed above. In brief, the enzyme catalyzes the
formation of an indolyloxazoline intermediate that undergoes either nonenzymatic isomerization leading to an
,
-dehydro
product, or hydration to form
-hydroxy and then
-keto products. More recently, another enzyme was reported to
catalyze the formation of an
,
-dehydrocysteinyl residue
during the biosynthesis of epidermin, a 52-residue peptide antibiotic
produced by Staphylococcus epidermidis Tü3298(17) . Post-translational
modification of the precursor peptide leads to the formation of the
unusual S-[(Z)-2-aminovinyl]-D-cysteine
residue at the C-terminal position of epidermin, involving the
oxidative decarboxylation of a C-terminal L-cysteine residue.
A two-step reaction is therefore anticipated, involving the formation
of an
,
-dehydrocysteine residue catalyzed by a new
flavoprotein EpiD (17) , and then followed by subsequent
decarboxylation. The mechanism of this reaction and the role of EpiD in
the decarboxylation reaction remain to be elucidated. However, the
reactional mechanism of EpiD is also clearly distinct from that of L-tryptophan 2`,3`-oxidase in that it has been recently
demonstrated to exhibit a strict substrate specificity toward peptides
of at least 4 residues possessing close sequence analogies to the
C-terminal epidermin tetrapeptide(18) . Presumably, a variety
of still unknown enzymes may cause dehydrogenation of amino acids,
hence contributing to their metabolic fate.