Conformational changes in orotidine 5′-monophosphate decarboxylase: "remote" residues that stabilize the active conformation

Article abstract of DOI:10.1021/bi100443a

The structural factors responsible for the extraordinary rate enhancement (～10¹⁷) of the reaction catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) have not been defined. Catalysis requires a conformational change that closes an active site loop and "clamps" the orotate base proximal to hydrogen-bonded networks that destabilize the substrate and stabilize the intermediate. In the OMPDC from Methanobacter thermoautotrophicus, a "remote" structurally conserved cluster of hydrophobic residues that includes Val 182 in the active site loop is assembled in the closed, catalytically active conformation. Substitution of these residues with Ala decreases k_cat/K_m with a minimal effect on k_cat, providing evidence that the cluster stabilizes the closed conformation. The intrinsic binding energies of the 5′-phosphate group of orotidine 5′-monophosphate for the mutant enzymes are similar to that for the wild type, supporting this conclusion.

Full text of DOI:10.1021/bi100443a

3
514 Biochemistry 2010, 49, 3514–3516
DOI: 10.1021/bi100443a
0
Conformational Changes in Orotidine 5 -Monophosphate Decarboxylase:
†
“
Remote” Residues That Stabilize the Active Conformation
‡
§
‡
B. McKay Wood, Tina L. Amyes, Alexander A. Fedorov, Elena V. Fedorov, Andrew Shabila, Steven C. Almo,
§
,‡
John P. Richard, and John A. Gerlt*
‡
§
Departments of Biochemistry and Chemistry, University of Illinois, Urbana, Illinois 61801, Department of Chemistry,
University at Buffalo, Buffalo, New York 14260, and Department of Biochemistry, Albert Einstein College of Medicine, Bronx,
New York 10461
Received March 24, 2010; Revised Manuscript Received April 5, 2010
ABSTRACT: The structural factors responsible for the extra-
(2) The kcat/K for OMP exceeds that for 1-(β-D-erythrofuranosyl)-
m
17
0
ordinary rate enhancement (∼10 ) of the reaction catalyzed
orotic acid (EO; 5 -truncated OMP analogue) by factors of 5.2 ꢀ
0
8
8
by orotidine 5 -monophosphate decarboxylase (OMPDC)
10 and 3.6 ꢀ 10 for ScOMPDC and MtOMPDC, respec-
have not been defined. Catalysis requires a conformational
change that closes an active site loop and “clamps” the
orotate base proximal to hydrogen-bonded networks that
destabilize the substrate and stabilize the intermediate. In
the OMPDC from Methanobacter thermoautotrophicus, a
tively (11, 12). (3) Phosphite dianion (HP ) activates decarboxy-
i
5
5
-1
lation of EO by factors of 5.6 ꢀ 10 and 2.9 ꢀ 10 M for
ScOMPDC and MtOMPDC, respectively (11, 12). The “intrinsic
0
binding energy” [IBE (13)] of the 5 -phosphate/HP (1) increases the
i
affinity for the substrate (e.g., OMP vs orotidine/EO) and (2)
enables decarboxylation by juxtaposition of the substrate with the
active site hydrogen-bonded networks (substrate destabilization
and intermediate stabilization). How the IBE promotes catalysis is
unknown but required to understand the structural basis for the rate
enhancement.
“remote” structurally conserved cluster of hydrophobic
residues that includes Val 182 in the active site loop is
assembled in the closed, catalytically active conformation.
Substitution of these residues with Ala decreases kcat/K with
m
a minimal effect on kcat, providing evidence that the cluster
stabilizes the closed conformation. The intrinsic binding
A loop located at the end of the seventh β-strand closes over
the active site when OMP binds (Figure 1). Although the active
site loops differ in both length and sequence in divergent
0
0
energies of the 5 -phosphate group of orotidine 5 -monophos-
phate for the mutant enzymes are similar to that for the wild
type, supporting this conclusion.
OMPDCs (12), each includes a spatially conserved Gln (Gln
0
1
85 in MtOMPDC) hydrogen-bonded to both the 5 -phosphate
and O2 of the pyrimidine as well as a conserved Ser at the end of
the fifth β-strand that also is hydrogen-bonded to N3 of the
0
1
Orotidine 5 -monophosphate decarboxylase (OMPDC) is one
of Nature’s best catalysts: the reaction occurs with a rate
17
23
enhancement of ∼10 and a proficiency of ∼10 M (2). The
reaction coordinate includes a vinyl carbanion intermediate
(
Scheme 1) (3, 4).
Structures of OMPDCs in complexes with UMP or 6-hydroxy-
UMP, an intermediate analogue (5-9), reveal hydrogen-bonded
networks proximal to (1) C6 of the pyrimidine [Asp 70-Lys 72-
Asp 75 in the OMPDC from Methanobacter thermoautotrophicus
(MtOMPDC)] and (2) O2, N3, and O4 of the base (Ser 127-
Gln 185). These destabilize the substrate (9) and stabilize the
intermediate, although the structural strategy for the latter is
unknown.
0
The 5 -phosphate group binds in a conserved motif at the ends
of the seventh and eighth β-strands of the (β/R) -barrel structure,
8
with interactions to backbone NH groups as well as the guani-
dinium group of a conserved Arg (Arg 203 in MtOMPDC).
These interactions are important for catalysis. (1) The k /K for
OMP exceeds that for orotidine by a factor of ∼10 for the
OMPDC from Saccharomyces cerevisiae (ScOMPDC) (10).
F
IGURE 1: Superposition of the active site of wild-type MtOMPDC
cat
m
in the absence (cyan; disordered loop depicted with the dotted line)
and presence (gray) of 6-hydroxyUMP. The carbons of 6-hydroxy-
UMP are colored yellow; the carbons of Val 182 and Arg 203 in the
liganded structure are colored orange.
1
1
†
This work was supported by National Institutes of Health Grants
Scheme 1
GM039754 to J.P.R. and GM065155 to J.A.G.
*To whom correspondence should be addressed. Telephone: (217)
2
44-7414. Fax: (217) 244-6538. E-mail: j-gerlt@uiuc.edu.
1
0
Abbreviations: OMP, orotidine 5 -monophosphate; OMPDC, OMP
decarboxylase; MtOMPDC, OMPDC from Methanobacter thermoau-
totrophicus; ScOMPDC, OMPDC from Saccharomyces cerevisiae; EO,
1-(β-
D
-erythrofuranosyl)orotic acid; HP
i
, phosphate dianion; IBE, in-
trinsic binding energy.
pubs.acs.org/Biochemistry
Published on Web 04/06/2010
r 2010 American Chemical Society

Rapid Report
Biochemistry, Vol. 49, No. 17, 2010 3515
0
Table 1: Kinetic Constants for OMP, EO, and EO HP and Intrinsic Binding Energies of the 5 -Phosphate Group of OMP at pH 7.1 and 25 °C
i
3
q
q
q
0
(
k
cat
)
OMP
(kcat/K
m
)
OMP
ΔΔG
(kcat/K
m
)
EO
ΔΔG
(kcat/K
m
)/
ΔΔG
5 -phosphate
-
1
)
-1 -1
a
-1 -1
a
b
-2 -1
a
c
MtOMPDC
(s
(M
s
)
(kcal/mol)
(M
s
)
(kcal/mol)
(K
D
)
EO HPi (M
s
)
(kcal/mol)
IBE (kcal/mol)
3
6
-3
-3
d
wild type
V182A
4.6
3.4
3.9
4.0
3.3
3.1
2.5
2.9 ꢀ 10
1.4 ꢀ 10
9.1 ꢀ 10
9.5 ꢀ 10
2.8 ꢀ 10
4.9 ꢀ 10
4.9 ꢀ 10
8.7 ꢀ 10
2500
190
980
690
340
81
11.6
5
1.8
0.7
0.7
1.4
2.4
2.4
1.3 ꢀ 10
1.1
0.9
0.6
0.8
1.8
1.7
1.5
0.6
0.8
1.2
2.0
2.6
10.9
11.8
11.5
11.0
11.0
10.9
5
5
5
4
4
-3
I199A
1.9 ꢀ 10
3.1 ꢀ 10
-3
-3
V201A
I218A
2.3 ꢀ 10
-4
V182A/I199A
V182A/V201A
a
3.9 ꢀ 10
5.0 ꢀ 10
-4
30
b
Calculated from the ratio of the second-order or third-order rate constants for the wild-type and mutant enzyme. Third-order rate constant for reaction of
c
0
EO HP
i
.
Transition state stabilization by the 5 -phosphate group of OMP, calculated from the ratio of the values of kcat/K
phosphite dianion can be calculated from the ratio of (kcat/K
for OMP is partially diffusion-controlled (1).
m
for OMP and EO. IBEs for
for EO. This value is a lower estimate because the value of kcat/K
3
d
m
)/K
D
for EO HP
i
and kcat/K
m
m
3
pyrimidine (Ser 127). We characterized the importance of these
clamp” residues in ScOMPDC (Gln 215-Ser 154) using EO HP
“
3
i
0
and confirmed that the 5 -phosphate HP “switch” is required to
activate the enzyme (14).
3
i
The structures of divergent OMPDCs reveal that OMP binding
is always accompanied by a conformational change (Figure 1).
The most obvious component is closure of the active site loop.
However, the (β/R) -barrel structure can be divided into two
8
domains, one formed from the second, third, fourth, and fifth
β-strands (where the hydrogen-bonded Asp 70-Lys 72-Asp 75
motif and Ser 127 are located) and the second from the sixth,
seventh, eighth, and first β-strands (where the phosphate binding
motif and the active site loop, including Gln 185, are located) (15).
OMP binding reorients the domains, with the latter domain
moving toward the former, forcing the orotate carboxylate group
to be juxtaposed vis- ꢀa -vis the Asp 70-Lys 72-Asp 75 motif and,
also, allowing formation of the Ser 127-Gln 185 clamp. Thus, the
transition between the open and closed conformations is more
complicated than “simple” hinge motion of the loop on the rigid
framework of the (β/R) -barrel structure. In this work, we identify
8
“
remote” residues involved in this conformational change and
F
IGURE 2: Superpositions ofthe 6-hydroxyUMP-liganded structures
quantitate their importance in promoting and stabilizing the
catalytically competent form of the enzyme.
of wild-type MtOMPDC and the single mutants in the hydrophobic
cluster: (A) hydrophobic cluster and (B) active sites.
The active site loop of MtOMPDC, P180-G181-V182-
G183-A184-Q185-G186-G187-D188, is disordered in the ab-
sence of substrate but ordered and closed in its presence
Scheme 2
(
Figure 1). In the liganded structure, two residues in the loop
make contacts with the (β/R) -barrel scaffold. (1) Gln 185 is
8
hydrogen-bonded to Ser 127 (vide infra), and (2) Val 182 is
embedded in a hydrophobic cluster also formed by Ile 199, Val
2
01, and Ile 218. The structural conservation of this hydro-
phobic cluster in all OMPDCs suggests its importance in a
common catalytic strategy. We probed this strategy by muta-
genesis of these hydrophobic residues.
Instead, the effects can be explained by decreased stabilities of
the closed conformation in which the substrate is destabilized (9)
and the anionic intermediate is stabilized. A consistent model
Ala substitutions for residues in the hydrophobic cluster cause
substantial decreases in kcat/K for decarboxylation of OMP but
m
have little effect on kcat (Table 1). We determined high-resolution
X-ray structures (e1.4 A) for each mutant in the presence of
(Scheme 2) is one in which an equilibrium of open (E ) and closed
o
˚
(E ) conformations exists in the absence or presence of the
c
0
0
6-hydroxyUMP (Figure 2). The liganded structures superimpose
substrate (Kc or K ; K , 1). The substitutions destabilize Ec
c
c
0
(decrease Kc and K ), so kcat/Km is decreased (the energy
c
well with that of the wild type, with only small differences
observed at the sites of the substitutions (panel A). The active
sites are identical to that of the wild type (panel B), explaining the
q
difference between E þ S and [E S] is increased). However,
o
c
3
the invariance of k establishes that the substitutions do not
cat
minimal impact on kcat
The mutated residues are remote from the active site (Figures 1
and 2). Thus, the effects of the substitutions on kcat/K cannot be
explained by altered direct interactions with the substrate.
.
alter the reactivitiy of E S (the energy difference between E S
c
3
c
3
q
and [E S] ). The energy to form E S from E and S is derived
c c o
3
3
0
from (1) interactions of the 5 -phosphate group with its binding
motif (IBE; vide infra) and (2) an increased concentration of
m

3
516 Biochemistry, Vol. 49, No. 17, 2010
Wood et al.
SUPPORTING INFORMATION AVAILABLE
0
0
s
OMP to form E S (K /K or, equivalently, K /K , although the
former is expected to be the relevant pathway).
We also used the two-part EO HP substrate. The values of
c
3
c
s
c
Descriptions of the experimental procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
3
i
kcat/Km for EO are decreased relative to that for the wild type
(
(
Table 1); these can be explained by decreased populations of E
Scheme 2), assuming that EO, without a 5 -phosphate group, is
c
0
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o
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3
i
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6
0
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1
1
1
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