COMMUNICATION
Cryo-crystallography of a True Substrate, Indole-3-glycerol
Phosphate, Bound to a Mutant (
D60N) Tryptophan Synthase
2
2 Complex Reveals the Correct
Orientation of Active Site
Glu49*
Sangkee
Rhee
,
Edith Wilson
Miles§, and
David R.
Davies
¶
From the
Laboratory of Molecular Biology and the
§ Laboratory of Biochemistry and Genetics, NIDDK, National
Institutes of Health, Bethesda, Maryland 20892
 |
ABSTRACT |
The reversible cleavage of indole-3-glycerol by
the
-subunit of tryptophan synthase has been proposed to be
catalyzed by
Glu49 and
Asp60. Although previous x-ray
crystallographic structures of the tryptophan synthase
2
2 complex showed an interaction between
the carboxylate of
Asp60 and the bound inhibitor indole-3-propanol phosphate, the carboxylate of
Glu49 was too distant to play its proposed role. To clarify the structural and functional roles of
Glu49, we have determined crystal structures of a mutant (
D60N)
2
2 complex in the presence and absence of
the true substrate, indole-3-glycerol phosphate. The enzyme in the
crystal cleaves indole-3-glycerol phosphate very slowly at room
temperature but not under cryo-conditions of 95 K. The structure of the
complex with the true substrate obtained by cryo-crystallography
reveals that indole-3-glycerol phosphate and indole-3-propanol
phosphate have similar binding modes but different torsion angles. Most importantly, the side chain of
Glu49 interacts with 3-hydroxyl group
of indole-3-glycerol phosphate as proposed. The movement of the side
chain of
Glu49 into an extended conformation upon binding the true
substrate provides evidence for an induced fit mechanism. Our results
demonstrate how cryo-crystallography and mutagenesis can provide
insight into enzyme mechanism.
 |
INTRODUCTION |
Various biochemical and physical methods, including site-directed
mutagenesis, kinetic analysis, and x-ray crystallography, have revealed
many mechanistic aspects of the bifunctional bacterial tryptophan
synthase
2
2 complex (EC 4.2.1.20) (for
reviews see Refs. 1 and 2). These studies provide evidence that
-subunit residues Asp60 and Glu49 catalyze the reversible cleavage of IGP1 to glyceraldehyde
3-phosphate and indole (
-reaction; see Scheme 1) (3-6). Recent
x-ray crystallographic studies, however, demonstrated that whereas the
side chain of
Asp60 interacts with the bound inhibitor IPP, the side
chain of
Glu49 is too distant (~6 Å) from IPP to play its
proposed role (7). However, IPP lacks the hydroxyl groups of the true
substrate IGP, and this could affect the orientation of
Glu49. To
investigate interactions between active site residues of the
-subunit and IGP, we have formed the enzyme-IGP complex using the
mutant (
D60N)
2
2 complex, in which the
other catalytic residue
Asp60 is replaced with Asn. This enzyme has
no measurable activity in the reaction catalyzed by the
-subunit but
retains substantial
-subunit activity (5). Here we present crystal
structures obtained by cryo-crystallography of the mutant (
D60N)
tryptophan synthase in the presence and absence of the bound true
substrate IGP.
 |
EXPERIMENTAL PROCEDURES |
The expression and purification of the mutant (
D60N)
tryptophan synthase
2
2 complex from
Salmonella typhimurium has been described (5). Although four
different substitutions were made at position 60, only the
D60N
2
2 complex yielded crystals suitable for
further study. Crystals of the
D60N
2
2
complex were grown under the conditions used previously for
crystallization of the wild-type enzyme (50 mM
N,N-bis(2-hydroxyethyl)glycine, 1 mM
Na-EDTA, 0.8-1.5 mM spermine, and 12% polyethylene glycol
8000 adjusted to pH 7.8 with NaOH) (8) and belong to the space group
C2.
D60N crystals grown in the presence of Na+ were
soaked for 1-2 days in a standard K+ soaking solution
containing 100 mM
N,N-bis(2-hydroxyethyl)glycine (pH 7.8 titrated
with KOH), 1 mM EDTA, and 20% polyethylene glycol 8000 (9), and these K+-soaked crystals were used for further
soaking experiments with ligands (see below).
Preliminary x-ray diffraction data collected at room temperature from
IGP-soaked
D60N crystals indicated that there was no electron
density for IGP bound to the
-active sites of the enzyme and
suggested that the crystalline enzyme still catalyzes the slow cleavage
of IGP. Therefore, to eliminate low enzymatic activity of crystalline
tryptophan synthase, we have flash frozen substrate-bound
D60N
crystals and then collected diffraction data at 95 K.
Substrate IGP was prepared enzymatically with tryptophan synthase from
indole and glyceraldehyde 3-phosphate (10). IGP was introduced into
D60N crystals by soaking the crystals in a IGP soaking solution (0.4 mM IGP with the standard solution) for 1 day. The crystals
were then transferred to a IGP soaking solution for 30 min into each of
a series of solutions having 5, 10, 15, 20, and 25% glycerol as
cryoprotectant and then were flash frozen for data collection. To
evaluate the effects of the
D60N mutation on the structure, the
unliganded
D60N crystals prepared as above were also flash frozen
and subjected to data collection. Diffraction data were collected at 95 K on a Raxis IIC imaging plate system mounted on a Rigaku RU-200
rotating anode x-ray generator operating at 50 kV and 100 mA. All
diffraction data were integrated with DENZO and scaled with SCALEPACK
(11). Table I summarizes data statistics
and refinement statistics. In refining these structures with X-PLOR
(12), the 2.0 Å wild-type structure determined in the presence of
K+ at room temperature (Protein Data Bank entry 1TTQ) (9)
served as a starting model. The starting model was divided into three substructures (corresponding to the
-subunit, and N- and C-terminal domains of the
-subunits) and subject to a rigid body refinement followed by simulated annealing refinement. Subsequent manual rebuilding was carried out using the program O (13). At this stage,
Fo
Fc maps of
D60N-IGP revealed the bound IGP in the
-subunit. An idealized
model of IGP was modeled using QUANTA and fitted to the density then
refined again by simulated annealing refinement followed by positional
and temperature factor refinements.
 |
RESULTS AND DISCUSSION |
Glu49 Interacts with the True Substrate IGP--
Structural
comparisons indicate that
D60N and the starting model (the wild-type
structure) are almost identical within root mean square deviation of
0.52 Å for the main chain atoms of all residues, suggesting that the
mutation does not produce any significant structural perturbations.
Subsequent comparisons between
D60N and
D60N-IGP also indicated
that there are no noticeable conformational changes induced by the
binding of IGP. The
D60N-IGP structure is similar to that of the
wild type-IPP complex (14) in that loop 6 (residues 179-191) is
invisible and loop 2 (residues 53-62 including residue 60) is highly
disordered.
Fig. 1A shows the electron
density map for IGP and nearby residues in the
D60N-IGP structure.
The overall binding site is almost identical to that of other
structures complexed with IPP that have been determined previously (7,
14). However, the
D60N-IGP structure reveals that the side chain of
Glu49 adopts an extended conformation and is within 2.8 Å from the
hydroxyl group of C3' of IGP (Fig. 1B). This interaction has
been proposed in the mechanism of
-reaction (Scheme
1) but has not been seen in other
structures with the bound substrate analog IPP (7, 14). The positions
of Asn60 in the
D60N-IGP structure is almost identical to that of
the IPP-complexed structure, but Asn60 is very mobile (refined
temperature factors for the all atoms in Asn60 are about 80 Å2).

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Fig. 1.
A, the final 2Fo Fc map overlaid on the models of IGP and
residues Glu49 and Tyr175 in the D60N-IGP complex. The map was
contoured at 0.6 . B, superposition between IGP
(open circles) in the D60N-IGP and IPP (filled
circles) in the K87T-Ser-IPP complex (7). The carboxylate of
Asp60 is shown near the indole nitrogen of IPP and of IGP.
|
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Scheme 1.
Proposed mechanism of the reaction. The cleavage of indole-3-glycerol phosphate (I) is
activated by tautomerization of the indole ring to yield the indolenine
tautomer (II), which undergoes aldol cleavage to yield III. These steps
are catalyzed by three putative residues, B1,
B2, and B3 (5). The binding geometry of
indole-3-propanol phosphate suggests that Asp60 may serve as
B2 residue, which abstracts the proton on N-1 of the indole
ring or forms a strong hydrogen bond to polarize the nitrogen atom.
Glu49 is thought to serve as B3, which accepts the
proton on the hydroxyl group. Glu49 may also serve as B1
that protonates the C3 position of the indole moiety. (Reproduced with
permission from Ref. 2).
|
|
Despite the similarity in the overall binding site, there are
differences in positions (0.9-1.4 Å) of the corresponding atoms including phosphate, C1', C2', and C3' but not in positions of indole
ring (Fig. 1B). The torsion angle around the C2'-C3' bond is
169 ° for IGP but 28 ° for IPP. Because there is relatively weak
electron density for the C1' atom of IGP, we initially modeled IGP
according to the torsion angles of IPP. This fitting keeps the hydroxyl
group at C2' away from its corresponding density, suggesting that the
current C1' atom represents the reliable position in IGP. Interaction
between
Glu49 and the C3' hydroxyl group is unambiguous based on
clear density. The C2' hydroxyl group is in D-enantiomeric
configuration but does not form any hydrogen bonds with active site
residues.
Other Active Site Residues around Bound IGP--
Fig. 1 also shows
that the phenolic hydroxyl of
Tyr175 interacts with the C3' hydroxyl
group of IGP. However, the finding that the mutant enzyme in which
Tyr175 is replaced by Phe (Y175F) has substantial activity indicates
that
Tyr175 is not essential for catalysis or substrate binding (5).
Early studies showed that whereas the Y175C mutant was inactive, a
second site revertant (Y175C/G211E) exhibited partial activity in the
-reaction (15). Later biochemical investigations of this mutant
enzyme combined with computer graphics modeling of the substrate
binding site of the
-subunit (5) led to the conclusion that the
partial restoration of the
-subunit activity in the doubly altered
second site revertant results from restoration of the proper geometry of the substrate binding site. This conclusion supports the view that
Tyr
175 serves a structural role but is not an essential
catalytic residue.
Catalytic Role of
-Subunit
Glu49--
The structure of the
inactive mutant (
D60N) with bound IGP reveals two new structural
features that were not observed in structures in the presence of
substrate analog IPP. These features are the interaction between
Glu49 and the hydroxyl group at C3' and the location of the
hydroxyl groups at C2' and C3'.
There is substantial evidence from site-directed mutagenesis studies
that
Glu49 (4, 6) and
Asp60 (5) are catalytic bases in the
reaction catalyzed by the
-subunit. The mechanism of this reaction
(Scheme 1), based on previous proposals (3-5), suggested that
Asp60
facilitates tautomerization of the indole ring of IGP (I) to form II
and that
Glu49 abstracts a proton from the C3' hydroxyl group of the
glycerolphosphate moiety to form III.
Although previous x-ray crystallographic structures of the tryptophan
synthase
2
2 complex demonstrated
interaction between the carboxylate of
Asp60 and the indole nitrogen
of the bound inhibitor (IPP), the side chain of
Glu49 was too
distant to play its proposed role (7). The carboxylate of
Glu49 was
folded away from the IPP and was located approximately 6.1 Å from the modeled C3' hydroxyl group (Fig. 1B). In the new structure,
the side chain of
Glu49 interacts with the C3' hydroxyl group of IGP
as proposed. This interaction is made possible by the movement of the
side chain of
Glu49 into an extended form in the presence of IGP.
These results provide an example of an induced fit mechanism in which
an active site residue adopts a catalytically correct orientation when
a substrate is bound to the active site. This type of induced fit is
much smaller and more localized than the larger changes observed in
some other structures which involve domain movement, loop closure, or
conversion from an open to a closed conformation.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The atomic coordinates and structure factors (code 1a5a for
D6ON and 1a5b for
D6ON-IGP) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
¶
To whom correspondence should be addressed: NIH, Bldg. 5, Rm.
338, Bethesda, MD 20892. Tel.: 301-496-4295; Fax: 301-496-0201.
1
The abbreviations used are: IGP,
indole-3-glycerol phosphate; IPP, indole-3-propanol phosphate.
 |
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