Mechanistic Studies on Tryptophan Lyase (NosL): Identification of Cyanide as a Reaction Product

Article abstract of DOI:10.1021/jacs.7b09000

Tryptophan lyase (NosL) catalyzes the formation of 3-methylindole-2-carboxylic acid and 3-methylindole from l-tryptophan. In this paper, we provide evidence supporting a formate radical intermediate and demonstrate that cyanide is a byproduct of the NosL-catalyzed reaction with l-tryptophan. These experiments require a major revision of the NosL mechanism and uncover an unanticipated connection between NosL and HydG, the radical SAM enzyme that forms cyanide and carbon monoxide from tyrosine during the biosynthesis of the metallo-cluster of the [Fe-Fe] hydrogenase.

Full text of DOI:10.1021/jacs.7b09000

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Communication
Mechanistic Studies on Tryptophan Lyase (NosL):
Identification of Cyanide as a Reaction Product
Dhananjay M Bhandari, Dmytro Fedoseyenko, and Tadhg P. Begley
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b09000 • Publication Date (Web): 12 Dec 2017
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Mechanistic Studies on Tryptophan Lyase (NosL): Identification of
Cyanide as a Reaction Product
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Dhananjay M. Bhandari, Dmytro Fedoseyenkoand Tadhg P. Begley*
Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States
Supporting Information Placeholder
supported by substrate analog studies and the recent de-
tection of 16 by EPR.10-11 In this communication, we
will describe three new experiments that support a varia-
tion of Mechanism B.
ABSTRACT: Tryptophan lyase catalyzes the formation
of 3-methylindole-2-carboxylic acid and 3-methylindole
from L-tryptophan. In this paper, we provide evidence
supporting a formate radical intermediate and demon-
strate that cyanide is a byproduct of the NosL-catalyzed
reaction with L-tryptophan. These experiments require a
major revision of the NosL mechanism and uncover an
unanticipated connection between NosL and HydG – the
radical SAM enzyme that forms cyanide and carbon
monoxide from tyrosine during the biosynthesis of the
metallo-cluster of the [Fe-Fe] hydrogenase.
O
O
NH3
N
H
OH
H
H
NH2
1
3
4
2
Path A
Path B
2
OH
3
4’
7’
NosL
3’
3a’
5’
6’
O
SAM, e-
2’
N
H
1’
7a’
O
1
NH3
OH
N
H
O
Tryptophan lyase (NosL) catalyzes the complex rear-
rangement of L-tryptophan (1) to 3-methylindole-2-
carboxylic acid (2), a component of the nosiheptide an-
tibiotic (Figure 1).1 This enzyme is a radical SAM en-
zyme in the same family of aromatic amino acid lyases
as ThiH (thiamin biosynthesis), HydG (Fe-Fe hydrogen-
ase maturation) and CofH (F420 biosynthesis).2 Recent
structural3 and biochemical studies4-5 have demonstrated
5
6
4
Figure 1: The NosL-catalyzed reaction as currently un-
derstood. Ammonia, formaldehyde and glyoxal are the
assumed byproducts.
H
H2O
N
O
O
H
5
NH3
Ÿ
that the 5′-deoxyadenosyl radical (Ado ) of NosL ab-
HO
4
NH
O
6
stracts a hydrogen atom from the amino group of L-
tryptophan (1) and that the enzyme shows remarkable
substrate tolerance.6-8 The current mechanistic proposals
for this reaction are shown in Figure 2. In mechanism
A,4, 6, 9 cleavage of the C2-C3 bond generates 8. Radical
addition to the carboxylate carbonyl of 9 gives 10. A
second β-scission reaction then gives 11, which tautom-
erizes to the final product 2. Reduction of the
methanimine radical (12) by the [4Fe-4S]+1 cluster fol-
lowed by hydrolysis completes the reaction. As a side
product 3-methylindole (5) is also produced in this reac-
tion at levels that are sensitive to the reaction conditions
and the substrate structure.1, 4 In particular, 5 is the ma-
jor product formed by the dithionite reduced enzyme
(ratio of 5:2 is ~ 95:05). A mechanistic explanation for
the control of the 2:5 ratio is not yet known. In Mecha-
nism B, β-scission of 7 generates the formate radical
(15) which then adds to C2' of 14 to give 16. A second
β-scission gives 11 and 12 which are converted to prod-
ucts as shown for mechanism A. Mechanism A is sup-
ported by substrate analog studies while Mechanism B is
HO
OH
O
HO
O
NH
N
H
N
Mechanism A
N
H
H
7
NH
8
10
9
OH
OH
O
N
H
O
N
H
11
2
NH
H
NH
H
O
H
H2O
NH3
H
H
4
3
13
12
NH
NH
O
Mechanism B
O
OH
O
N
H
N
H
14
15
16
Figure 2: Two proposed mechanisms for the NosL-
catalyzed reactions.
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Replacing the tryptophan carboxylate with a thiocar-
resulting reaction mixture showed a new signal at 24.3
min (Ex = 270 nm, Em = 352 nm) that comigrated with
an authentic sample of 22 after NaBH4 reduction. LC-
MS analysis confirmed the identification of this product
as the aldehyde 21 (see Figure S5).
1
2
3
4
5
6
7
8
boxylate is expected to favor formation of 20 over 5 if
the reaction proceeds via Mechanism A because sulfur
will stabilize 10 (OH bond is 18 kcal/mole stronger than
the SH bond). In contrast, for Mechanism B, this sub-
strate modification will retard the indole carboxylation
because of thioformate radical (19) stabilization by the
sulfur. This will facilitate the trapping of 14. When the
NosL-catalyzed reaction was performed with tryptophan
thiocarboxylate (17), HPLC analysis of the reaction
mixture showed complete substrate consumption (Figure
3) and formation of a new product, which was identified
as aldehyde 21 based on LC-MS analysis (Figure S4).
This product was also reduced with NaBH4 to form al-
cohol 22, which was chromatographically identical with
an authentic standard. Remarkably, 3-methylindole (5),
the major product formed from tryptophan, was not de-
SH
N
H
O
N
H
20
5
9
NH
NH2
O
NH
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
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SH
SH
NosL
S
N
H
N
H
N
H
O
14
19
17
18
Ado
Ado-H
- NH3
H2O
O
OH
NaBH4
tected.
N
H
N
H
NH2
22
21
SH
O
Figure 4: Mechanistic proposal for the NosL catalyzed
reaction with L-tryptophan thiocarboxylate (17). The
thioformate radical (19) is converted to carbon dioxide
(SI, Page 19).
N
H
O
Ado-H
17
N
H
21
2'-Halo-L-tryptophan (Br, Cl) was designed to trap a
radical on C3′ by exploiting the very rapid cleavage of a
C-halogen bond beta to a radical center.14-15 When the
NosL reaction was carried out with 2'-chloro-L-
tryptophan (23), HPLC analysis showed complete sub-
strate consumption and exclusive formation of a new
product (Figure 5). This product was identified as 3-
methylindole-2-carboxylic acid (2) by LC-MS and
chromatographic identity to an authentic standard (see
Figure S7). Similar results were obtained with 2'-bromo-
L-tryptophan (24, see Figure S8). This is the first time
we have observed the exclusive formation of 2 in a
NosL-catalyzed reaction and demonstrates that trypto-
phan halogenation at C2 is one possible strategy for con-
trolling the 2:5 ratio.
MTA
Time (min)
Figure 3: HPLC analysis of the NosL-catalyzed reaction
with L-tryptophan thiocarboxylate (17). Ado-H, 5′-
deoxyadenosine; MTA, 5′-methylthioadenosine (SAM
degradation product).
A mechanistic proposal for this reaction is shown in
Figure 4. In this proposal β-scission of the C1-C2 bond
of 18 generates 14 and the thioformate radical (19).
Imine hydrolysis gives 21. Remarkably this reaction
does not yield any 3-methylindole (5) suggesting that
addition of the formate radical to the indole precedes
and is required for cleavage of the C2-C3 bond. Failure
of the thioformate radical (19) to add to the indole is
likely to be a consequence of the extra stabilization of
the radical by delocalization into an empty d-orbital of
sulfur (approx. 10 kcal/mole).12 In addition, the weaker
hydrogen bonding of Arg323 to sulfur is likely to lower
the efficiency of delivering thioformate (19) to the in-
dole.
NH2
OH
OH
O
Cl
N
H
O
2
N
H
23
Ado-H
MTA
We have previously observed the formation of alde-
hyde 14 in the reaction of D-tryptophan with NosL but
viewed this fragmentation as an off-pathway reaction
because of the altered tryptophan stereochemistry.4, 13
Aldehyde 14 however was not previously detected from
L-tryptophan. Motivated by the results of the tryptophan
thiocarboxylate experiment, we repeated the NosL/L-
tryptophan reaction using fluorescence rather than ab-
sorbance for product detection to give greater sensitivity
for the detection of aldehyde 21. HPLC analysis of the
Time (min)
Figure 5: HPLC analysis of NosL reaction with 2'-
chloro-L-tryptophan (23). The peak at 20 min in the full
reaction
mixture
corresponds
to
5′-deoxy-5′-
thioadenosine whose formation has been previously de-
scribed.7
2
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This reaction mixture was next analyzed for the prod-
methylindole (5) is the major heterocyclic product of
this reaction4 and glyoxal (6) formaldehyde and ammo-
nia (4) were assumed to be the C1/C2, the C2 and C2
amino-derived products respectively, based on the detec-
tion, at trace levels by LC-MS, of glyoxal (6) and for-
maldehyde in the reaction mixture (Figure 1).1 The de-
tection of cyanide as a NosL reaction product in the 2'-
chloro-L-tryptophan (23) reaction cast doubt on this as-
sumption.
1
2
3
4
5
6
7
8
ucts derived from C2 and the C2 amino group of 2'-
chloro-L-tryptophan (23). Formaldehyde, carbon mon-
oxide and cyanide were each considered as possible
products. The enzymatic reaction, treated with purpald
or dansylhydrazine failed to yield the corresponding
formaldehyde adducts, thus excluding formaldehyde as a
byproduct. Reduced hemoglobin also failed to detect
carbon monoxide.16 To test for cyanide formation, the
enzymatic reaction was treated with orthonapthalene
dicarboxaldehyde (25) and taurine (26) to convert cya-
nide to the fluorescent derivative 28 (Figure 6).17 HPLC
analysis using fluorescence (Ex = 418 nm, Em = 454
nm)18 showed a new product confirmed as 28 by LC-MS
and chromatographic identity with an authentic standard
(Figure S10). The ratio of cyanide to 3-methylindole-2-
carboxylic acid was ~ 0.9:1.
9
A)
NH
NH2
NH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
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40
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44
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OH
OH
H
NosL
O
O
Cl
O
Cl
Cl
N
N
H
N
H
30
O
H
23
Ado
15
29
Ado-H
H
H
N
N
NH
H
C
C
H
H
OH
OH
OH
O
A)
N
O
N
H
N
H
O
O
Cl
Cl
N
H
C
SO3
Cl
32’
32
31
H2N
H
H
26
N
N
SO3
N
HC
H
25
C
28
Ex = 418 nm, Em = 454 nm
O
27
H
B)
OH
O
OH
O
- Cl
N
N
H
N
H
Cl
C
2
33
N
B)
SO3
NH2
O
H
28
OH
NosL
OH
HCN
27
N
H
N
H
N
H
O
Cl
Ado
Ado-H
34
35
Figure 7: A) Mechanistic proposal for the NosL-
catalyzed reaction of 2'-chloro-L-tryptophan (23). B)
2,3-dimethyl indole (35) and cyanide are the products
generated during the NosL-catalyzed reaction with 2'-
methyl- L-tryptophan (34).
Time (min)
Figure 6: Detection of cyanide as a byproduct of the
NosL-catalyzed reaction. A) Reaction used to convert
cyanide to a fluorescent product. B) HPLC analysis of
the NosL/2'-chloro-L-tryptophan (23) reaction mixture
after derivatization as shown in panel A.
When the NosL reaction was performed with [1,2,3-
13C3, 2-amino-15N]-L-tryptophan (prepared from labeled
serine and indole using tryptophan synthase19), 13C-
NMR analysis of the reaction mixture revealed only two
new signals (8 ppm and 160 ppm, Figure 8A). The 8
ppm signal was assigned to the methyl group of 3-
methylindole (5) and the 160 ppm signal was assigned
to cyanide and/or bicarbonate by comparison with the
spectra of authentic standards in reaction buffer.
A mechanistic proposal for this reaction is shown in
Figure 7A. Hydrogen atom abstraction from the amino
group of 2'-chloro-L-tryptophan gives 29. A β-scission
reaction releases the formate radical (15), which then
adds to C2' of the indole to give 31. Loss of H2CN+ from
31 gives the stabilized radical anion 32 which can also
be viewed as an aryl radical anion 32'. Protonation at
C3, potentially using the strongly acidic H2CN+, fol-
lowed by loss of a chlorine atom completes the reaction.
This proposal suggests that replacing the chloride with a
methyl group would result in the formation of 2,3-
dimethylindole (35) due to rapid decarboxylation of the
methyl analog of intermediate 33. In fact, this is the ob-
served product of 2'-methyl-L-tryptophan (34) with
NosL (see Figures 7B and S11-S12).
Since the 160 ppm signal could not be unambiguously
identified, we repeated the experiment using [1-13C1]-L-
tryptophan. 13C-NMR analysis of the resulting reaction
mixture showed a single signal at 160 ppm that was
identical to that of a spiked bicarbonate standard demon-
strating that the carboxy group of tryptophan is convert-
ed to CO2 (see Figures S14-S15). This conclusion was
further verified by demonstrating CO2 production using
phosphoenolpyruvate carboxylase (see Figures S16-
S19). To determine if cyanide is also a reaction product,
the reaction mixture was treated with orthonapthalene
dicarboxaldehyde (25) and taurine (26) (Figure 6).
HPLC and LC-MS analysis of this reaction mixture
Next, we turned our attention to the identification of
the products of dithionite-reduced NosL with L-
tryptophan (1). We previously established that 3-
3
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demonstrated the formation of the fluorescent product
28 ([M-H]- = 299 Da). When identical reactions were
carried out using [15N]-L-tryptophan and [1,2,3-13C3, 2-
amino-15N]-L-tryptophan, the masses of the fluorescent
products ([M-H]-) were 300 Da and 301 Da respectively
(see Figures S20-S21). This is consistent with the con-
version of C2 and the C2 amino group of L-tryptophan to
cyanide (3-methylindole:cyanide ratio ~ 1:0.9).
oxide from tyrosine during the biosynthesis of the metal-
lo-cluster of the [Fe-Fe] hydrogenase.20
1
2
3
4
5
6
7
8
NH
NH2
NH
OH
OH
NosL
O
O
O
N
H
N
H
N
H
O
7
15
1
Ado
14
Ado-H
H
H
N
N
NH
CH
CH
OH
9
OH
OH
A)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
H
O
N
H
O
N
H
O
36’
36
16
-
CN
H
O
O
O
N
H
O
15
3*
H
37
B)
15NH2
- e-
H
H
H
3
*
1
*
OH
2
*
1*
O
O
O
O
H
O
2*
N
H
N
H
N
H
H
N
H
CO2
5
38
2
Figure 9: Mechanistic proposal for the NosL-catalyzed
reaction that is consistent with the formation of cyanide
from the C2 and C2 amino group of L-tryptophan.
Figure 8: A) 13C-NMR of the NosL-catalyzed reaction
with [1,2,3-13C3, 2-amino-15N]-L-tryptophan. B) 13C-
NMR of [1,2,3-13C3, 2-amino-15N]-L-tryptophan stand-
ard.
Funding Sources
This research was supported by the Robert A. Welch Foun-
dation (A-0034).
These results can be integrated into the mechanistic
proposal for the NosL-catalyzed reaction with L-
tryptophan (1) shown in Figure 9. In this mechanism,
the initially formed amine radical (7) undergoes a β-
scission reaction to form 14 and the formate radical (15)
which then adds to the C2' of the indole to form 16. Loss
of H2CN+ by a second β-scission reaction gives 36
which can also be described as an aryl radical anion 36'.
Protonation of this radical by the strongly acidic H2CN+
followed by electron transfer back to the [4Fe-4S]+2
cluster gives the cation 38. This can lose CO2 or a pro-
ton to give the experimentally observed reaction prod-
ucts 5 and 2 respectively. Alternatively, loss of a for-
mate radical from 37 could also give 5. This mechanism
is consistent with the EPR spectroscopy that provides
evidence for the formation of 16. It is also consistent
with the NosL reaction with L-tryptophan thiocarbox-
ylate (17), which blocks the formation of 16 and demon-
strates that the formation of 3-methylindole (5) can only
occur after addition of the formate radical to the indole.
Finally, it is consistent with the formation of 3-
methylindole-2-carboxylic acid (2) as the sole product
when 2'-chloro-L-tryptophan (23) is used as the substrate
and with the formation of 2,3-dimethyl indole (35) when
2'-methyl-L-tryptophan (34) is used as the substrate. The
identification of cyanide as the product derived from the
C2 and C2 amino group of tryptophan requires a major
revision of the NosL mechanism and uncovers an unan-
ticipated connection between NosL and HydG – the rad-
ical SAM enzyme that forms cyanide and carbon mon-
Supporting Information.
The Supporting Information is available free of charge on
the ACS Publications website at DOI:
Detailed procedures for substrate synthesis, chemoen-
zymatic synthesis, enzymatic reaction conditions, HPLC
and LC-MS chromatograms of all enzymatic reaction
products, and NMR spectra of synthesized compounds are
provided.
AUTHOR INFORMATION
Corresponding Author
*begley@chem.tamu.edu
Phone: (979) 862-4091.
ORCID
Dhananjay M. Bhandari: 0000-0003-4250-3303
Dmytro Fedoseyenko: 0000-0001-7776-8278
Tadhg P. Begley: 0000-0001-5134-2623
ACKNOWLEDGMENT
Ron Trolard, Cambridge Isotope Laboratories, provided
[13C3,15N1]-L-serine as a generous gift.
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NH2
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CN
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H
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Ado
Ado-H
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