The pyridine ring of NAD is formed by a nonenzymatic pericyclic reaction

Article abstract of DOI:10.1021/ja0446395

The biosynthesis of quinolinate 3, the precursor to the pyridine ring of NAD, is still poorly understood. Two pathways have been identified, one involving the direct formation of quinolinic acid from aspartate and dihydroxyacetone phosphate, the other requiring a five-step degradation of tryptophan. The final step in this degradation is catalyzed by the non-heme Fe(II)-dependent enzyme 3-hydroxyanthranilate-3,4-dioxygenase (HAD). This enzyme catalyzes the oxidative ring opening of 3-hydroxyanthranilate (1) to 2-amino-3-carboxymuconic semialdehyde (ACMS, 2) which then cyclizes to quinolinate (3). In this communication, we demonstrate the following: (1) cyclization of ACMS to 3 is not HAD catalyzed, (2) the most stable form of ACMS in solution is an all trans isomer which undergoes facile cis to trans isomerization about the C2-C3 and C4-C5 double bonds via transient formation of its enol tautomer (6), (3) a model study on the ring opening of N,N-dimethylcarbamoylpyridinium with hydroxide and methoxide suggests that the cyclization of ACMS occurs by an electrocyclization reaction of its enol tautomer 6. Thus, the biosynthesis of quinolinic acid, by the tryptophan pathway, is likely to be a member of a growing family of natural products whose biosynthesis involves a pericyclic reaction. Copyright

Full text of DOI:10.1021/ja0446395

Published on Web 12/24/2004
The Pyridine Ring of NAD Is Formed by a Nonenzymatic Pericyclic Reaction
Keri L. Colabroy and Tadhg P. Begley*
Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell UniVersity, Ithaca, New York 14850
Received September 3, 2004; E-mail: tpb2@cornell.edu
Scheme 1
The biosynthesis of quinolinate 3, the precursor to the pyridine
ring of NAD, is still poorly understood. Two pathways have been
identified, one involving the direct formation of quinolinic acid
from aspartate and dihydroxyacetone phosphate,1 the other requiring
a five-step degradation of tryptophan.2 The final step in this
degradation is catalyzed by the non-heme Fe(II)-dependent enzyme
3-hydroxyanthranilate-3,4-dioxygenase (HAD). This enzyme cata-
lyzes the oxidative ring opening of 3-hydroxyanthranilate (1) to
2-amino-3-carboxymuconic semialdehyde (ACMS, 2) which then
cyclizes to quinolinate (3). Until recently, quinolinate formation
from 1 was difficult to study because HAD was an unstable, low-
abundance protein in all sources examined.3-6 However, recent
identification and characterization of the stable Ralstonia metalli-
durans HAD, which can be overexpressed at a high level in
Escherichia coli, has greatly facilitated the mechanistic character-
ization of this system.2 In this communication we describe the
solution structure of the most stable isomer of 2 and the mechanisms
for its isomerization and conversion to 3.
Scheme 2
Previous studies, using low concentrations of partially purified
beef kidney HAD to generate ACMS, suggested that the cyclization
of ACMS (2) to quinolinate (3) is not enzyme catalyzed.7 We have
confirmed this by treating HAD-free ACMS, readily prepared by
the enzymatic oxidation of 1 followed by chloroform precipitation
and ultrafiltration, with high concentrations of active, purified HAD.
The “enzymatic” and nonenzymatic cyclization rates were 0.011
min-1 and 0.015 min-1, respectively. While this experiment clearly
demonstrates that the cyclization of ACMS is not catalyzed by
HAD, it does not rule out the possibility that a separate, as yet
uncharacterized protein, catalyzes this reaction.
Scheme 3
The conversion of 2 to quinolinate involves isomerization to 4
(see below) followed by cyclization to 7 and dehydration to quino-
linate 3 (Scheme 2). Two mechanisms have been proposed for the
cyclization reaction.7 In mechanism A, the amine undergoes a nu-
cleophilic addition to the aldehyde to give 5. This mechanism poses
several problems: (1) the amine lone pair is poorly oriented for
overlap with the antibonding orbital of the aldehyde, (2) this amine
is a weak nucleophile having the reactivity of a vinylogous amide
rather than that of an amine, and (3) electron donation from the
amine to the aldehyde carbon attenuates the electrophilicity of this
carbon, making it more like an amide than an aldehyde carbonyl.
This analysis is supported by the low reactivity of ACMS (2) with
strong nucleophiles such as hydroxylamine and by the low basicity
of the amine (pKa < 5).7 On the basis of these considerations, we
expect that cyclization of 4 by the nucleophilic addition mechanism
is likely to be very slow. An alternative that overcomes these prob-
lems is shown in mechanism B in which 4 tautomerizes to 6, which
can then undergo a 6π electrocyclization reaction to give 7. Previous
efforts to differentiate between these mechanisms using isotope
effects, pH-rate studies and acid-base catalysis were unsuccessful.7
N,N-Dimethylcarbamoylpyridinium (8) undergoes facile ring
opening to give a product assumed to be 12.8,9 This reaction involves
the addition of hydroxide to the pyridinium ring followed by ring
opening. This ring opening is a model for the microscopic reverse of
the conversion of 4 to 7. It should be possible to differentiate
between ring opening by an elimination (9 to 10 in Scheme 3sthe
reverse of 4 to 5 in Scheme 2) and ring opening by a pericyclic
reaction (9 to 11 via mechanism B in Scheme 3sthe reverse of 6
to 7 in Scheme 2) by comparing the rates of the hydroxide- and
methoxide-mediated reactions. For hydroxide, facile ring opening
could occur by both mechanisms, while for methoxide, ring opening
by mechanism A is expected to be much slower than ring opening
by mechanism B because methoxy is a much weaker electron donor
than alkoxy.
When N,N-dimethylcarbamoylpyridinium 8 was treated with
hydroxide (0.7 equiv) in D2O, rapid ring opening occurred. NMR
analysis of the reaction mixture demonstrated the formation of two
products 14 and 15, which were easily differentiated based on the
chemical shifts of the aldehydic and the corresponding enol protons
9
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J. AM. CHEM. SOC. 2005, 127, 840-841
10.1021/ja0446395 CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Scheme 4
Figure 1. NMR analysis of the time course for the HAD-catalyzed
conversion of 3-hydroxyanthranilate (1) to quinolinate (3). After 30 min
peaks due to a single isomer of ACMS were evident; after 96 min, most of
the substrate was consumed, and peaks due to both ACMS and quinolinate
were evident. After 23 h, all of the substrate was converted to quinolinate.
at 9.04 and 7.54 ppm, respectively (Scheme 4). Treatment of N,N-
dimethylcarbamoylpyridinium 8 with methoxide (0.7 equiv) in
CD3OD also resulted in rapid ring opening. NMR analysis of this
reaction mixture demonstrated the formation of 16 as the major
product (Scheme 4). The chemical shift of the C5 vinyl proton of
16 in D2O (7.53 ppm) was consistent with the corresponding
assignment of the C5 proton of 15 (7.54 ppm). In both cases
complete reaction occurred within the mixing time, and no rate
difference between the two reactions was evident. This suggests
that the ring opening of 9 is occurring by a pericyclic reaction and
supports the hypothesis that the conversion of 4 to 7 is also
occurring by a pericyclic reaction.
Scheme 5
The coupling constants for H1-H5 for compounds 14, 15, and 16
are consistent with the trans stereochemistry for each of the CC
double bonds, suggesting that the initially formed cis isomer 12
undergoes facile rotation about the C1-C2 and the C3-C4 bonds.
To determine the solution structure of ACMS (2), it was generated
by the HAD-catalyzed oxidation of 1 in D2O buffer, and the reaction
mixture was analyzed by NMR (Figure 1). This analysis demonstra-
ted that ACMS was formed as a single isomer (Figure 1). This iso-
mer is likely to be the aldehyde rather than the enol tautomer be-
cause the chemical shift of the C6 proton (8.8 ppm) is closer to that
of the C5 proton of 14 (9.04 ppm) than the C5 proton of 15 (7.54
ppm) or 16 (7.53 ppm). The coupling constants between the H4, H5,
and H6 protons of ACMS are consistent with a trans C4-C5 double
bond. This was confirmed by the observation of an NOE between
H4 and H6. The 1H NMR analysis does not enable us to assign the
stereochemistry about the C2-C3 double bond of ACMS. However,
since both carboxylates are ionized at pH ) 7 and ACMS is confor-
mationally mobile, it is likely that these groups are trans to minimize
electrostatic repulsion. We therefore suggest that the all trans isomer
of ACMS (17) represents the most stable stereoisomer in solution.
The all trans stereoisomer of ACMS (17) must undergo two cis/
trans isomerization reactions, one about the C2-C3 double bond,
the other about the C4-C5 double bond, before it can undergo the
cyclization reaction to give 7. These isomerization reactions do not
involve ACMS protonation at C5 because ACMS does not undergo
H/D exchange with buffer and no solvent deuterium is incorporated
into the quinolinic acid. (Figure 1). We therefore suggest that ACMS
(17) is in equilibrium with a small concentration of its enol tautomer
18 and that the required double bond rotations occur from this
tautomer (Scheme 5). This is consistent with the observations that
12 rapidly isomerizes to 14 and that 14 is in equilibrium with 15
(Schemes 3 and 4).
isomer 17 is the most stable form of ACMS in solution and that
17 undergoes facile cis to trans isomerization about the C2-C3
and C4-C5 double bonds via transient formation of its enol tauto-
mer 18. A model study on the ring opening of dimethylcarbamoyl-
pyridinium with hydroxide and methoxide suggests that the cycliza-
tion of ACMS occurs by an electrocyclization reaction of the enol
tautomer 6. Thus, the biosynthesis of quinolinic acid by the trypto-
phan pathway is likely to be a member of a growing family of
natural products whose biosynthesis involves a pericyclic reaction.10
Acknowledgment. We thank Ivan Kerestes for help with the
NMR analysis and Barry Carpenter for helpful discussions. This
research was supported by a grant from NIH (DK44083).
Supporting Information Available: NMR spectra and experimen-
tal preparations of HAD, ACMS, 8, and 14-16. This material is
available free of charge via the Internet at http://www.pubs.acs.org.
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Our studies confirm that the conversion of ACMS to quinolinate
is not enzyme catalyzed. In addition, we propose that the all trans
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