Chemical models and their mechanistic implications for the transformation of 6-cyanouridine 5′-monophosphate catalyzed by orotidine 5′-monophosphate decarboxylase

Article abstract of DOI:10.1039/c001865a

The reactions of 6-cyano-1,3-dimethyluracil have been studied as chemical models to illustrate the mechanism for the transformation of 6-cyanouridine 5′-monophosphate (6-CN-UMP) to barbiturate ribonucleoside 5′-monophosphate (BMP) catalyzed by orotidine 5′-monophosphate decarboxylase (ODCase). The results suggest that the Asp residue in the ODCase active site plays the role of a general base in the transformation.

Full text of DOI:10.1039/c001865a

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Chemical models and their mechanistic implications for the
transformation of 6-cyanouridine 50-monophosphate catalyzed
by orotidine 50-monophosphate decarboxylasew
Yuen-Jen Wu, Chen-Chieh Liao, Cheng-Hung Jen, Yu-Chiao Shih and Tun-Cheng Chien*
Received 27th January 2010, Accepted 21st April 2010
First published as an Advance Article on the web 25th May 2010
DOI: 10.1039/c001865a
The reactions of 6-cyano-1,3-dimethyluracil have been studied as
chemical models to illustrate the mechanism for the trans-
formation of 6-cyanouridine 50-monophosphate (6-CN-UMP) to
barbiturate ribonucleoside 50-monophosphate (BMP) catalyzed
by orotidine 50-monophosphate decarboxylase (ODCase). The
results suggest that the Asp residue in the ODCase active site
plays the role of a general base in the transformation.
catalysis. These facts suggest that the proficient catalyst
operates by a novel chemical mechanism.3–6
In 2005, Kotra et al. discovered that 6-CN-UMP (3) under-
went hydrolysis to form BMP (4) in the active site of ODCase
from Methanobacterium thermoautotrophicum (MtODCase)
(Scheme 1(B)(a)).7 It was the first example that ODCase
catalyzes an alternative substrate to undergo a reaction other
than decarboxylation. We speculated that both the trans-
formation of 6-CN-UMP (3) and the decarboxylation of
OMP (1) (Scheme 1(A) & (B)(a)) would involve the same
or similar chemical actions. Understanding the chemical
reactions of 6-cyanouracil derivatives could open up an
opportunity for studying the enzymatic mechanism of ODCase.
The reactions of 6-cyanouracil derivatives reported by
Senda et al. in the 1970s8–10 have received our attention. When
6-cyano-1,3-dimethyluracil (6-CN-1,3-DMU, 7) was treated
with sodium methoxide, n-butylamine or hydrazine, the
6-cyano group was replaced by the incoming nucleophiles to
give the corresponding 6-substituted 1,3-dimethyluracils
(6-MeO-1,3-DMU (8), 6-n-BuNH-1,3-DMU (9) and
6-H2NNH-1,3-DMU (10), respectively). We envisioned that
the nucleophilic substitutions of 6-CN-1,3-DMU (7) could
potentially be a chemical model to account for the enzymatic
transformation of 6-CN-UMP (3).
ODCase (EC 4.1.1.23) catalyzes the decarboxylation of OMP
(1) to uridine 50-monophosphate (UMP, 2) in the final step of
the de novo pyrimidine nucleotide biosynthesis (Scheme 1(A)).
ODCase is one of the most proficient enzymes known.1,2
Unlike most of the other decarboxylases, ODCase contains
no metal ions or small molecule cofactors, and there is no
evidence for the existence of a covalent intermediate during the
We rationalized that the MtODCase-catalyzed trans-
formation of 6-CN-UMP (3) to BMP (4) could occur in two
possible pathways:11 (A) Direct hydrolysis: A water molecule
undergoes the nucleophilic substitution of the 6-cyano group
to form BMP (4) (Scheme 2(A)); (B) Covalent intermediate:
A nucleophilic residue undergoes the nucleophilic substitution
at the 6-position of 6-CN-UMP (3) to form a substrate-
enzyme covalent complex. Further hydrolysis of the covalent
intermediate accomplishes the transformation to form BMP (4)
Scheme 1
Department of Chemistry, National Taiwan Normal University,
No.88, Sec.4, Ting-Zhou Road, Taipei, 11677, Taiwan.
E-mail: tcchien@ntnu.edu.tw; Fax: +886 2-29324249;
Tel: +886 2-77346126
w A part of the work has been presented as a poster in the Joint
Symposium of the 18th International Roundtable on Nucleosides,
Nucleotides and Nucleic Acids and the 35th International Symposium
on Nucleic Acids Chemistry, Sep. 8–12, 2008, Kyoto, Japan.11
Scheme 2
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(Scheme 2(B)) (also proposed by Rudolph et al. in their recent
paper12,13). X-Ray crystallography and sequence alignments
have identified conserved amino acid residues in the active site
consisting of two lysines and two aspartates.14,15 The function
of the Asp–Lys–Asp–Lys tetrad remains unclear except for the
charge interactions and the formation of a hydrogen-bonding
network. Herein, we postulate that the Lys and/or the Asp will
participate in the catalytic reaction.
Alternatively, when 6-CN-1,3-DMU (7) was treated with
alkoxides in alcohols, nucleophilic substitution reactions took
place to give 6-alkoxy-1,3-dimethyluracils (8 & 16). This
direct substitution reaction of 6-CN-1,3-DMU (7) could be
considered as an ideal model for the enzymatic transformation
of 6-CN-UMP (3). To clarify the function of the Asp residue
in the transformation, acetate was used to mimic the Asp
residue in the model reactions. When 6-CN-1,3-DMU (7) was
heated in the presence of NaOAc in ethanol, 6-EtO-1,3-DMU
(16) was obtained in a good yield. The same result was found
with Na2CO3 in ethanol. A catalytic amount of NaOAc or
Na2CO3 in ethanol afforded the same product in comparable
yields. These results revealed that the ethanolysis of 6-CN-1,3-
DMU (7) is a general base catalyzed nucleophilic substitution
reaction and acetate plays the role of the general base in the
chemical reaction (Table 1, X = CN). Meanwhile, the reaction
of 6-Cl-1,3-DMU (15) was also exemplified as a chemical
model for the enzymatic transformation of 6-I-UMP (5).
Nucleophilic displacement with n-BuNH2 proceeded readily
to give 6-n-Bu-NH-1,3-DMU (9), which represented the
formation of a Lys-UMP adduct from 6-I-UMP (5) in the
active site of wild-type MtODCase. On the other hand,
subjecting 6-Cl-1,3-DMU (15) to NaOAc or Na2CO3 in
ethanol afforded 6-EtO-1,3-DMU (16) as the only product,
which suggests that the nucleophilic substitution is substan-
tially catalyzed by a general base as well (Table 1, X = Cl).
Kotra et al. recently reported the crystal structures for the
complexes of several MtODCase mutants with 6-CN-UMP (3)
and 6-I-UMP (5).17 The D70A and D75N mutants formed a
covalent bond between the C-6 position of UMP and the Lys
residue when these mutants were incubated with 6-CN-UMP
(3) (Scheme 1(B)(b)). This result indicated that, in the absence
of the Asp residue in the active site, the transformation of
6-CN-UMP (3) to BMP (4) could not proceed and that the Lys
side chain underwent the substitution reaction to form the
Lys-UMP complex instead (Scheme 1(B)(b)). In contrast,
In an effort to examine the covalent mechanism
(Scheme 2(B)) with the participation of the Lys residue,
6-CN-1,3-DMU (7) was used as the chemical model and was
reacted with n-butylamine to afford 6-n-Bu-NH-1,3-DMU (9)
in analogy to the formation of the substrate-enzyme covalent
intermediate. Hydrolysis of 6-n-Bu-NH-1,3-DMU (9) in an
aqueous HCl ethanol solution furnished the formation of
6-OH-1,3-DMU (11) (Scheme 3). This chemical model
suggests that the proposed covalent mechanism could be
feasible.12,13 However, Kotra et al. reported that 6-iodouridine
50-monophosphate (6-I-UMP, 5) irreversibly inhibited the
catalytic activities of MtODCase.16 The inhibitor was
covalently bound to the wild-type MtODCase between the
6-position of the uridine base and the Lys residue in the active
site (Scheme 1(B)(d)). Hydrolysis of the Lys-UMP complex to
BMP and free enzyme did not occur. In contrast, the covalent
adduct was not observed when 6-CN-UMP (3) was incubated
with the wild-type MtODCase, and the enzyme activity
was inhibited solely by BMP (4). Thus, based upon their
results, we have concluded that the transformation of
6-CN-UMP (3) to BMP (4) does not go through the
Lys-UMP complex (Scheme 2(B)).
In an attempt to learn if the enzymatic transformation of
6-CN-UMP (3) was due to the direct displacement of the
cyano group with H2O, 6-CN-1,3-DMU (7) was subjected to
the reactions with several oxygen-nucleophiles in protic and
aprotic solvents. These reactions gave a variety of products
including the migration of the cyano group from the
6-position to the 5-position,8–10 and the hydrolysis of the
cyano compounds to the corresponding amides (13 & 14)
(Scheme 4). Although the direct substitution of the cyano
group with a water molecule seems straightforward, the
desired product, 6-OH-1,3-DMU (11), was not observed.
Table 1 Reactions of 6-CN-1,3-DMU under nucleophilic conditions
Entry
X
Reagent (eq)
Solvent
Time/h Products (yield)
1
2
3
4
CN
n-BuNH2
MeOH
EtOH
3
1
1
1
9 (84%)
8 (64%)
CN NaOMe (1)
CN NaOEt (1)
CN NaOAc (2)
16 (73%)
16 (54%), 12a (5%),
17 (27%)
EtOH
Scheme 3
5
CN NaOAc (0.2) EtOH
CN Na2CO3 (2) EtOH
CN Na2CO3 (0.2) EtOH
1
16 (35%), 12a (2%),
17 (52%)
16 (75%)
6
7
8
9
1
1
18
24
16 (60%), 17 (26%)
9 (96%)
16 (8%),
recovered 15 (69%)
16 (96%)
Cl n-BuNH2 (2.2) DMF
Cl NaOAc (2)
Cl Na2CO3 (2)
EtOH
EtOH
10
28
a
5-CN-1,3-DMU (12).
Scheme 4
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This work was supported by Research Grant 96-2113-M-
003-004 from the National Science Council, Taiwan. We
are grateful to the National Center for High-performance
Computing of Taiwan for the electronic resources and facilities.
Notes and references
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Scheme 6
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without the Lys residue in the active site, the K72A mutant
transformed 6-I-UMP (5) to BMP (4) which is similar to how
6-CN-UMP (3) was hydrolyzed by the wild-type MtODCase
(Scheme 1(B)(c)).
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Our studies are in agreement with Kotra’s observations that
both 6-CN-1,3-DMU (7) and 6-Cl-1,3-DMU (15) underwent
ethanolysis in the presence of NaOAc, and that their reactions
with n-BuNH2 resulted in only 6-n-Bu-NH-1,3-DMU (9)
(summarized in Scheme 1(B) and Scheme 5). Furthermore,
in a competing experiment with a 1 : 1 mixture of both NaOAc
and n-BuNH2 in ethanol under reflux, to mimic the existence
of both Asp and Lys residues in the ODCase active site,
6-CN-1,3-DMU (7) only gave 6-EtO-1,3-DMU (16) while
6-Cl-1,3-DMU (15) predominantly afforded 6-n-Bu-NH-1,3-
DMU (9) (Scheme 6). The competing experiment has revealed
the distinct preferential reactivities of 6-CN-1,3-DMU (7) and
6-Cl-1,3-DMU (15), which could account for the different
transformations of 6-CN-UMP (3) and 6-I-UMP (5) catalyzed
by the same enzyme (Scheme 1(B), (a) & (d)).
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Our results from the chemical models may indicate that the
Asp residue in the active site could assume a role similar to
that of acetate in the chemical models. Thus, the model studies
suggest that the transformation of 6-CN-UMP (3) to BMP (4)
is catalyzed by MtODCase with the Asp residue as a general
base. While most of the model studies for ODCase have
focused on the explanation of transition state stablization
of the OMP decarboxylation,18–23 our chemical models have
established the role of the Asp residue in the ODCase active
site.24 It remains to be further investigated how the general
acid–base chemistry effects the decarboxylation of MtODCase.
23 P. Beak and B. Siegel, J. Am. Chem. Soc., 1976, 98, 3601–3606.
24 During the preparation of this manuscript, Rudolph et al. reported
that the cocrystallization of the human ODCase (hODCase)
with 6-CN-UMP (3) resulted in the presence of UMP (2) in the
active site, instead of BMP (4) ( ref. 13). The proposed mechanism
involved the hydrolysis of 6-CN-UMP (3) to OMP (1), followed by
a normal decarboxylation. In our model studies, we have observed
the formation of ethyl 1,3-dimethyluracil-6-carboxamidate (17) as
a by-product from the base-catalyzed ethanolysis (Table 1, entries
4, 5 and 7), and 1,3-dimethyluracil-6-carboxamide (13) as a result
of base-catalyzed hydrolysis. (Scheme 4) Accordingly, we antici-
pated that the Asp residue also plays the role of a general base for
the hODCase-catalyzed hydrolysis of 6-CN-UMP (3).
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This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 4821–4823 | 4823