(Received for publication, March 27, 1995; and in revised form, July 14, 1995)
From the
The roles of active site residues His,
Phe
, Lys
, and His
in the Streptomyces rubiginosusD-xylose isomerase were
probed by site-directed mutagenesis. The kinetic properties and crystal
structures of the mutant enzymes were characterized. The pH dependence
of diethylpyrocarbonate modification of His
suggests that
His
does not catalyze ring-opening as a general acid.
His
appears to be involved in anomeric selection and
stabilization of the acyclic transition state by hydrogen bonding.
Phe
stabilizes the acyclic-extended transition state
directly by hydrophobic interactions and/or indirectly by interactions
with Trp
and Phe
. Lys
and
His
mutants have little or no activity and the structures
of these mutants with D-xylose reveal cyclic
-D-xylopyranose. Lys
functions structurally
by maintaining the position of Pro
and Glu
and catalytically by interacting with acyclic-extended sugars.
His
provides structure for the M2-metal binding site with
properties which are necessary for extension and isomerization of the
substrate. A second M2 metal binding site (M2`) is observed at a
relatively lower occupancy when substrate is added consistent with the
hypothesis that the metal moves as the hydride is shifted on the
extended substrate
D-Xylose isomerase (EC 5.3.1.5) catalyzes the
reversible interconversions of D-xylose to D-xylulose
and D-glucose to D-fructose. The D-xylose
isomerase gene (xylA) has been cloned from a variety of
bacterial
sources(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) and
structures of the Streptomyces
rubiginosus(13, 14, 15) , Arthrobacter(16, 17, 18) , Actinoplanes missouriensis(19) , and Streptomyces
olivochromogenes(20, 21) enzymes have been
determined by x-ray crystallography. The catalytic domains fold as
eight-stranded /
barrel motifs and contain a well conserved
active site with two divalent metal ions(17) . All D-xylose isomerases require Mg
,
Co
, or Mn
ions for activity,
suggesting that they have similar enzymatic mechanisms.
Initially, a cis-enediol mechanism was proposed for D-xylose
isomerase(14, 22) , similar to the mechanism of
triose-phosphate isomerase. However, isotope exchange experiments (23) and crystallographic analyses (15, 16) with various substrates and inhibitors
suggests that the reaction proceeds via a metal-mediated hydride shift (Fig. 1a). The currently accepted pathway for the
reaction involves the preferential binding of
-D-xylopyranose (24, 25) followed by
ring opening(25) , extension of the substrate, and then the
hydride shift(15, 16, 26) . Recently, Meng et al.(27) have proposed that the hydride shift
occurs on the cyclic form of sugar (Fig. 1b).
Figure 1:
Cartoons of the metal-mediated hydride
shift (a) (15, 16, 26) and
cyclic-hydride transfer mechanisms (b)(27) . Residues
relevant to this study (His, Phe
,
Lys
, and His
) are shown, as well as
Asp
, which is important in the cyclic-hydride transfer
mechanism (b). Metal ions are labeled M1 and M2. In both
mechanisms the
-pyranose (D-xylose is shown here) binds
to the active site and after isomerization, the
-furanose form (D-xylulose is shown here) is released. In the metal-mediated
hydride shift, ring opening is required prior to isomerization, while
in the cyclic-hydride transfer ring opening and isomerization occur at
the same step.
Site-directed mutagenesis has been used to probe the functions of
specific active site residues in D-xylose
isomerase(27, 28, 29, 30, 31, 32) ,
however, only a few structures of mutant enzymes have been
reported(19, 33, 34, 35) . Kinetic
data can be misleading if the substitutions affect the properties of
catalytically important residues other than those changed by
mutagenesis. For this reason, we have shifted our mutagenic studies
from the Escherichia coliD-xylose
isomerase(28, 29) , which has not been successfully
crystallized, to the S. rubiginosus enzyme which readily forms
crystals diffracting x-rays beyond 2.0
Å(15, 34, 36) . Here, we report the
cloning and expression of S. rubiginosus xylA in E.
coli, and investigate the roles of His and Phe
in anomeric recognition, the putative role of His
in
ring opening, and the functions of His
, Phe
,
Lys
, and His
in isomerization.
Figure 2:
Electron densities for D-xylose
isomerase complexes with ligands. For all of the panels, residues are
labeled at CA, metal ions (asterisks) are labeled M1 and M2,
and water molecules (asterisks) are labeled Wat. The dashed lines depict metal ligation and hydrogen bonds which
have distances 3.2 Å and reasonable geometry. Cross-hatched lines represent electron density of the F
- F
``omit'' map. The contour level is 3
in panels a and b, and 4
in panels c-f. a, wild-type/THG. THG was modeled in two conformations (ALT1
and ALT2) and each was refined at 0.5 occupancy. The main difference in
ALT1 and ALT2 of THG is the position of O6. In ALT1, O6 is 2.8 Å
from Thr
OG, and in ALT2, O6 is 2.5 Å from
Wat
. The B factors for both models of THG, M1, and M2 are
17, 10, and 7 Å
, respectively. Although not indicated
in this figure, the positions of both metals shift 0.6 Å compared
to the wild-type structure without THG. The protein side chains
liganded to M1 shift 0.3 to 0.8 Å as a result of the new position
of the metal and also because of the proximity of the sugar hydroxyls
O3 and O4. Shifts in the positions of side chains liganded to M2 are
also observed, most likely because the ligands to M1 are repositioned.
The indole group of Trp
rotated 9° about
and the phenyl ring of Phe
(*, from a neighboring
subunit) rotated 5° about
when THG was added.
The amino side chain of Lys
moves 0.5 Å in order to
accommodate the new position of Wat
which is hydrogen
bonded to O2 of THG. His
NE2 is 2.9 Å from the ring
sulfur and 3.5 Å from the anomeric hydroxyl, and both O3 and O4
are liganded to M1 at 2.3 Å. b,
His
-Ser/xylose. D-Xylose was placed in the
electron density, but not added during refinement because the electron
density of the sugar is weak and in the absence of sugar, water
molecules are located close to the positions of the sugar hydroxyls and
would complicate an accurate determination of sugar occupancy (not
shown). Sugar binding would be comparable to a wild type-xylulose
complex(36) , except there is no hydrogen bond between
Ser
and O5. Instead, O5 of sugar could hydrogen bond to
Wat
and Wat
, a water not observed in a
wild-type/xylulose structure(36) . No water or metal were added
to the electron density near M2, although we suspect that this electron
density represents an alternate position for M2 (see Fig. 5). c, Lys
-Met/xylose.
-D-Xylopyranose
was modeled and refined in Lys
-Met. The B factors for the
sugar, M1, and M2 were 16, 12, and 13 Å
,
respectively. The binding of
-D-xylopyranose is
comparable to wild-type/THG, except
-D-xylopyranose lacks
C6 and O6. The sugar hydroxyls O3 and O4 are ligated to M1 at 2.4 and
2.3 Å, respectively. d, His
-Glu/xylose.
-D-Xylopyranose was modeled and refined in
His
-Glu. The B factor for the sugar is 13
Å
. Both metals are observed and metal ligation is
similar in the absence of sugar(34) , except M1 is liganded by
O3 and O4 of sugar at 2.4 and 2.2 Å, respectively. Glu
does not ligand M2, but can interact with O3 of sugar at 3.0
Å. e, His
-Ser/xylose.
-D-Xylopyranose was modeled and refined in
His
-Glu. The B factor for the sugar is 14
Å
. Both metals are observed and metal ligation is
similar to that observed in the absence of sugar(34) , except
M1 is liganded by O3 and O4 of sugar at 2.3 and 2.2 Å,
respectively. Two water molecules (Wat
and
Wat
) are located near Ser
and one water
(Wat
) replaces His
NE2 as a ligand to M2. f, His
-Ser/xylitol. The orientation of xylitol
is such that O1 interacts with Lys
NZ and O5 hydrogen
bonds to His
NE2. M1 is liganded by O2 and O4 at 2.2 and
2.3 Å, respectively. The B factor for xylitol is 6
Å
. M2 is absent, alternate conformations (ALT1 and
ALT2) for the side chain of Asp
and two new water
molecules (Wat
and Wat
) are shown.
Additional differences are shown and described in Fig. 7.
Figure 5:
Metal liganding at M2 (a) and M2` (b) in His-Ser/xylose. Labeling of metals,
residues, and water molecules are as described in the legend to Fig. 2. Metal ligation is depicted as dashed lines and
metal ligand distances are shown in Å. The ligation shown for M2`
is highly speculative because the ligands may move when the metal
shifts to the M2` position but due to the low occupancy at this site,
alternative ligand positions would not be observed in the electron
density. Wat
is close to the M2` site (1.5 Å (b)), but it is shown liganded to M2` because it may move when
M2 moves to M2`. The Asp
and Asp
carboxylate oxygens are 3.6 and 4.4 Å, respectively, from
M2`, and are not M2` ligands unless they move
correspondingly.
Figure 7:
Overlay of His-Ser in the
presence (bold lines) and absence (thin lines) of
xylitol, showing the perturbed structure caused by xylitol binding.
Labeling of water molecules, metal, and protein residues are as Fig. 2, except the five water molecules present in the absence
of xylitol and located near the xylitol hydroxyls in
His
-Ser/xylitol are not labeled. No M2, two alternative
positions (ALT1 and ALT2) for both the side chains of Glu
and Asp
, and two new water molecules (Wat
and Wat
which are shown in Fig. 2e)
are observed in His
-Ser/xylitol. The electron density of
the alternative conformations for both Asp
and
Glu
in the His
-Ser/xylitol structure
appeared to be equivalent (not shown), and each of the conformations
were modeled at 0.5 occupancy. The average B factors for ALT1 and ALT2
of the Asp
side chain are 13 and 8 Å
,
respectively, and for the two positions of the Glu
side
chain are 13 and 8 Å
. The new positions for the
Glu
side chain cause the phenyl group of Phe
to rotate 9° about
from its position
observed in the xylitol-free His
-Ser structure.
Significant movement of M1 (0.5 Å) Asp
OD2 (1.2
Å), and Asp
OD1 (0.6 Å, not shown) is also
observed, but M1 ligand distances (2.1 to 2.3 Å) do not differ
significantly from the previously described structures in this study or
others(15, 36) .
Figure 3: Activity of D-xylose isomerase and DEPC inactivation as a function of pH. Activity and enzyme inactivation are as described under ``Materials and Methods.''
Figure 4:
Anomeric preference of wild-type (),
Phe
-Ser (
), and His
-Asn (
) D-xylose isomerase. Crystalline
-D-xylopyranose
(96%) was dissolved, aliquots were removed, and the relative rate on
the mutarotating sugar was assayed at different time intervals by the
coupled-sorbitol dehydrogenase assay, as described under
``Materials and Methods.''
There is significant, unaccounted for electron density 3.5 Å from M1 and 1.9 Å from M2 (Fig. 2b). Since metal cannot occupy both the modeled position for M2 and the position at the excess density, it appears that this is an alternate site for M2. An equivalent, low occupancy site for M2 (M2`) has been described for the native S. rubiginosusD-xylose isomerase complexed with D-xylose (15) and is observed in our wild-type D-xylose complex. The M2` position has been proposed to stabilize the transition state of the acyclic-extended sugar in the metal-mediated hydride shift mechanism through interactions with O1 and O2 of sugar(15) .
In our wild-type structure, the relative difference peak heights
calculated from a F - F
map with M2 and M2` omitted is 0.12 e/A
for
M2 and 0.0092 e/A
for M2`. This yields an
estimated M2:M2` ratio of 13:1. Since the occupancy of M2` is 13-fold
lower and side chain atoms and water molecules have fewer electrons
than Mn
, low occupancy positions for the ligands of
M2` would not be seen above the noise in the electron density, as noted
by others (15) . The potential liganding of the metal at M2` by
Wat
, His
NE2, both carboxylate oxygens of
Glu
, and O1 and O2 of sugar is shown in Fig. 5.
Figure 6:
Overlay of the wild-type D-xylose
isomerase (thin lines) and Lys-Met (bold
lines) showing the perturbed structure around the Met
substitution. Labeling of water molecules, metal, and protein residues
are as described in the legend to Fig. 2. Hydrogen bonds from
Lys
NZ to Asp
OD 1, Glu
O,
and Wat
in the wild-type enzyme are shown by dashed
lines along with the corresponding distances for these
interactions. In wild-type and Lys
-Met,
Glu
, Pro
, and Arg
are the i, i+1, and i+2 residues in a
non-standard turn. In wild-type, Pro
is in a cis conformation while in Lys
-Met, Pro
is
in a trans conformation. The
/
angles for
Glu
, Pro
, and Arg
are 76/107
and -81/-16 and -84/158, respectively, while in
Lys
-Met, the angles are 116/-65,
-53/-33, and -135/160. The electron density around
the Met
side chain (not shown) is well ordered, and the
average B factor for the Met
side chain is 7
Å
. There are 0.7-2.0 Å shifts in the
positions of Glu
and Pro
. The new
conformation of Glu
in Lys
-Met is
stabilized by two new interactions, Glu
OE1-Asp
N and Pro
O-Arg
NH1 (not shown). The
new position of the main chain oxygen of Pro
results in
the loss of two hydrogen bonds from Arg
NH1 to two water
molecules (not shown) in Lys
-Met. The Phe
side chain rotates approximately 18° toward Met
CE when the polar amino group of Lys
is replaced by
the smaller non-polar Met, and Trp
rotates approximately
10° about
, as a result of Phe
rotation and the new position of Pro
. There are
also slight shifts (0.3-0.4 Å) in the positions of M1,
Glu
, and Asp
from that observed in the
wild-type structure (see Fig. 2e).
The loss of the
Lys-Glu
hydrogen bond and the increased van
der Waals radii associated with the Met side chain leads to the most
dramatic structural change, with the
Glu
-cis-Pro
peptide bond fliped
from the cis to the trans conformation (Fig. 6). The Glu
/
angles change from
76/107° to 116/-65, both of which are angles normally not
observed for non-glycine residues(51) . The energy of a Xaa-trans-Pro peptide bond is roughly 5-fold more
favorable than a Xaa-cis-Pro peptide
bond(51) .
The largest change observed when D-xylose is
added to His-Glu, is a 0.4-Å shift in the position
of Asp
OD. Glu
does not replace the
function of His
NE2 in serving as a ligand to M2 (Fig. 2d), as previously observed in
His
-Glu without sugar(34) , but can interact with
O3 at 3.0 Å. The position, coordination, and temperature factors
for both metals is similar in His
-Glu with or without D-xylose (34) .
The structure of
HisSer/xylose shows only slight rotation of Trp
and Phe
, but these positions are observed in the
wild-type/THG structure. There were no other significant changes
(>0.3 Å) in the positions of the metals or their ligands
compared to His
-Ser without sugar(34) . The two
new water molecules seen near the Ser
side chain,
Wat
and Wat
as well as the ligation of M2
by Wat
in His
-Ser/xylose (Fig. 2e), are also observed in His
-Ser
without D-xylose(34) .
Xylitol was added to His-Ser to mimic the
acyclic sugar binding typically seen in native D-xylose
isomerase-xylose complexes. The electron density of xylitol is well
defined and xylitol binds in an extended conformation (Fig. 2f). However, significant structural changes are
observed in and near the active site of His
-Ser/xylitol (Fig. 2f and Fig. 7). These changes include the
disappearance of M2, the addition of two new waters (Wat
and Wat
), alternative conformations for the side
chains Asp
and Glu
, rotation of
Phe
, and changes in the position of both M1 and its
carboxylate ligands. The electron density attributed to metal
(Mn
) at both M1 and M2 in all of the structures
described in this work can easily be seen at 12 to 15
. However,
in His
-Ser/xylitol only density around M1 is seen at 12
to 15
, and no peaks greater than 5
are observed around the
M2 site, strongly suggesting that M2 is not bound. In order to
accommodate O1 of xylitol, Wat
has moved 1.8 Å, and
is only 1.0 Å from the site once occupied by M2. Glu
OE2 shifted 1.2 Å and now hydrogen bonds to Wat
at 2.9 Å and to a new water molecule (Wat
) at
2.7 Å.
The S. rubiginosus xylA was cloned via PCR and
expressed in E. coli. The cloned PCR fragment contained
several nucleotide sequence differences, one of which gives rise to a
PheLeu mutation. In S. rubiginosusD-xylose isomerase, Phe
and the side chains
of Val
, Leu
, Phe
, and
Phe
are buried in a hydrophobic pocket, approximately 8.0
Å from the active site. Although we would not predict the
Leu
substitution to affect activity, it resulted in the
formation of insoluble inclusion bodies. After Leu
was
changed back to Phe, soluble, active D-xylose isomerase was
obtained, suggesting that the Phe
-Leu mutation caused
problems with protein folding.
While we do not have an alternative
explanation for the results of Meng et al.(27) the
data from our study and others are not readily consistent with the
cyclic-hydride transfer mechanism. First, Lys-Met,
His
-Ser, and His
-Glu complexed with D-xylose and wild-type/THG show that O2 is 4.5 Å from
the closest of the carboxylate oxygens (OD 2) of Asp
, and
that this carboxylate oxygen is liganded to M1. It is highly unlikely
that Asp
could be a base catalyst given the orientation
of the
-pyranose. Second, in the cyclic-hydride transfer
mechanism, M2 and Lys
would have no direct function in
catalysis, even though our biochemical and crystallographic data
indicate that both are important catalytically. The structure of
His
-Ser and His
-Asn do not show any major
structural perturbations at M1 or Asp
, yet these mutants
are almost totally inactive(34) . Structures of
His
-Ser, His
-Glu, and Lys
-Met
with D-xylose have
-D-xylopyranose in the active
site and display binding similar to wild-type/THG. Additional evidence
for a role of M2 in catalysis is shown by substitutions at residues
located near M2 ligands. Substitutions to Lys
(Lys
in S. rubiginosus) and Glu
change the metal specificity and pH profile of the A.
missouriensisD-xylose isomerase (30, 35) and structures of the Glu
-Gln
mutant with different metals show differences at the M2 site but not at
M1(35) . Lys
is essential for activity.
Replacement of Lys
with Met, Ser, Gln, or Arg renders the
enzyme inactive(30) . The structures of Lys
-Met
reveals no dramatic changes to M1, M2, or their ligands and is further
evidence against the cyclic-hydride transfer. However, the
Lys
-Met structures should be interpreted with caution
since other structural changes occur in this enzyme.
With the
exception of the results of Meng et al.(27) , the
aforementioned data and observations are consistent with the proposed
metal-mediated hydride shift mechanism (Fig. 1a)(15, 16) . When D-xylose, D-glucose, xylitol, or sorbitol are added
to D-xylose isomerase, an acyclic-extended form of sugar is
observed which may represent substrate, product, or intermediate(s)
(15, 16, 18, 19, 21, 26, 35, 36). The orientation of substrate is such
that O1 is hydrogen bonded to Lys NZ, O5 is hydrogen
bonded to His
NE2, and O2 and O4 of the extended sugar are
liganded to M1 (Fig. 1a). In the metal-mediated hydride
shift mechanism proposed by Collyer et al.(16) ,
Whitlow et al.(15) , and Lavie et
al.(26) , a M2 bound hydroxide initiates isomerization by
removal of the O2 hydrogen. M2 would shift approximately 1.9 Å to
a site where it could be liganded by both carboxylate oxygens of
Glu
, the imidazole NE2 of His
,
Wat
, and both O1 and O2 of substrate. The resulting
negative charge on O2 would be stabilized by both M1 and M2
interactions. The C2 hydrogen would transfer directly to the partial
positively-charged C1 and either a water molecule or Lys
NZ might protonate O1. Lys
NZ would help stabilize
the sugar by interacting with O1 of the acyclic-extended substrate,
intermediate(s), and product.
The results from this study and the
possible functions of His, Phe
,
Lys
, and His
in the reaction are discussed
below in terms of the metal-mediated hydride shift mechanism.
Phe does have a role in maintaining the structure of the active site,
sugar binding, and stabilization of the transition state. The structure
of Phe
-Ser shows two new waters and changes in the
positions of both the nearby hydrophobic side chains of Phe
and Trp
. Upon addition of D-xylose,
disordered density is observed in the active site, into which different
forms of sugar could be fit. As mentioned previously, the
Phe
-Ser mutation has a reduced k
and an increased K
(Table 1). These
results suggest that Phe
is involved in stabilization of
the acyclic extended transition state, through hydrophobic interactions
directly with the sugar or indirectly by interacting with the nearby
Trp
and Phe
side chains which in turn
contact the sugar.
After binding of the -pyranose, D-xylose isomerase catalyzes ring opening(25) . It was
suggested that His
NE2 could act as a catalytic base,
abstracting a proton from the anomeric oxygen (O1) of the
-pyranose, and facilitating sugar ring
cleavage(15, 16) . However, in the wild-type/THG
structure, His
NE2 is closer to the ring-sulfur of THG
(2.9 Å) than the anomeric hydroxyl (3.5 Å), suggesting that
the His
imidazole could act as a acid catalyst and thus
protonate the ring oxygen. The pH dependence of DEPC modification
indicates that the pK
of His
is 6.40
± 0.01. From kinetic studies of the
Mg
-activated ArthrobacterD-xylose
isomerase with fructose as a substrate, it was reported that the
pK
for a group controlling K
was 6.2 ± 0.1 and it was suggested that this group was
His
(His
in S.
rubiginosus)(53) . The pK
of
His
determined by DEPC inactivation is consistent with the
kinetic study of the Arthrobacter enzyme and indicates that at
the pH optimum of the enzyme (near 8.0-9.0), His
NE2
is deprotonated and probably could not be an acid catalyst in ring
opening. Changing His
to non-basic residues (Ser, Ala,
Asn, and Gln) reduces activity, but the rate-limiting step in the
overall reaction has been reported to be isomerization, not ring
opening(30, 31) . The acyclic sugar
5-deoxy-D-xylulose can be used as a substrate, indicating that
enzyme-catalyzed ring opening is not a step absolutely required prior
to isomerization. Other than crystallographic observations noting the
proximity of His
to cyclic sugars (15, 16) , there is no biochemical evidence suggesting
that His
catalyzes ring opening.
Initial site-directed
mutagenesis experiments of the E. coliD-xylose
isomerase suggested that His (His
in S.
rubiginosus) might be catalytically important in a cis-enediol mechanism, as replacement with Arg or Tyr rendered
the enzyme inactive(28) . However, when His
was
replaced with smaller residues (Ala, Ser, Asn, Asp, Glu, and Gln),
these mutant enzymes retained
activity(29, 30, 31) , proving that
His
is not crucial for catalysis and providing further
evidence against the cis-enediol mechanism.
His does, however, interact with the transition state. In most
crystal structures of D-xylose isomerase complexed with D-xylose, His
interacts with O5 of an
acyclic-extended form of sugar. Biochemical evidence for a
His
-sugar interaction is shown by comparing the kinetics
of the wild-type enzyme and His
mutants on D-xylose, D-xylulose, and 5-deoxy-D-xylulose (Table 2). Both mutants have lower k
and
higher K
values on D-xylose and D-xylulose when compared to the native enzyme. This is likely
due to the loss of a hydrogen bond from O5 of the extended sugar to the
substituted side chain. The interaction with the C5 hydrogen of
5-deoxy-D-xylulose and His
NE2 of the native
enzyme is clearly unfavorable, as reflected in both the diminished k
and elevated K
. A
possible reason for the His
mutants having a similar k
on D-xylulose and
5-deoxy-D-xylulose is that their smaller side chains cannot
hydrogen bond to O5 of D-xylulose, and the replaced side
chains are not close enough to interact unfavorably with the C5 methyl
group of 5-deoxy-D-xylulose.
Additional evidence for the
importance of the hydrogen bond from His to O5 of the
extended substrate can be inferred from the His
-Ser/xylose
structure. The Ser
side chain is 6.9 Å away from O5
of the extended sugar and thus cannot form a hydrogen bond to
substrate. The electron density contributed by the sugar in the active
site is weak and the temperature factors of the metals are high,
possibly because the hydrogen bond between substrate is lost and/or the
His
-Ser mutation makes the active site larger.
There are several possible reasons for observing
cyclic sugar in these mutants: (i) the extended form of sugar
represents product, (ii) the mutated side chains directly or indirectly
block the ring opening step, or (iii) the binding energy of cyclic (or
pseudo-cyclic) sugar is improved to the binding energy of the extended
substrate. Computer simulations of D-xylose isomerase with D-xylose estimated that there is an increase of 8 kcal/mol
when D-xylose goes from pseudo-cyclic (O3 and O4 ligated to
M1) to acyclic (O2 and O4 ligated to M1) conformations(55) .
Entropic contributions were not included in their calculation and might
lower this estimate. One possibility is that the acyclic form of sugar
observed bound to the wild-type D-xylose complexes is actually D-xylulose(15) . Since Lys-Met has no
activity, and His
-Ser and His
-Glu have only
0.3 and 0.5% activity (Table 1), respectively, it might be
expected that
-D-xylopyranose would be observed. However,
one S. olivochromogenesD-xylose isomerase mutant,
Glu
-Lys (Glu
in S. rubiginosus),
has no activity but shows an acyclic-extended conformation of D-glucose bound in the active site(33) . A direct role
for either Lys
or His
in ring opening is
unlikely since both are far (>7 Å) from the anomeric and ring
oxygens of substrate. We postulate that
-D-xylopyranose
is observed in the His
mutant structures because this
form is energetically more stable in the mutants than the
acyclic-extended conformation as M2 has dissociated from
His
-Ser/xylitol and the altered binding site cannot
provide the proper metal geometry and/or enough ligands. In the case of
Lys
-Met, we believe
-D-xylopyranose is
observed in the active site because the extended sugar is not
stabilized by the absent Lys
NZ-O1 sugar interaction and
thus the
-D-xylopyranose conformation becomes more
stable.
Lys could function catalytically in the
metal-mediated hydride shift by assisting in the polarization of O1 of
the transition state(15, 16, 30) .
Substituting the Lys side chain with Met rendered D-xylose
isomerase inactive. In studies of the A. missouriensisD-xylose isomerase, substitution with Ser, Gln, and Arg
also inactivated the enzyme(30) . The absence of activity may,
however, result from a structural defect that indirectly affects the
function of another catalytically important residue. The structures of
both Lys
-Met and Lys
-Met/xylose have large
perturbations at and around Glu
and Pro
.
Other changes are observed at Phe
and Trp
in the active site. Comparison of the wild-type enzyme complexed
with D-xylose (i.e. acyclic-extended sugar) to the
Lys
-Met-xylose complex suggests that the extended sugar
could be accommodated in the active site of Lys
-Met.
These results clearly indicate that Lys
is structurally
important but they also suggest that Lys
has a role in
extending the pseudo-cyclic sugar, stabilization of the
acyclic-extended sugar, and isomerization, by interacting with O1 of
the sugar.
His NE2 serves as a ligand to M2 when M2 is
in either the high occupancy site (M2) or the low occupancy site
(M2`)(15). Movement to the M2` may be concurrent with deprotonation of
the C2 hydroxy(56) . Perturbing the structure at M2 or M2`
could affect sugar extension by altering the properties of the metal(s)
or the metal-bound Wat
, both of which interact with O2
and/or O1 of the extended
sugar(15, 16, 19, 26, 36) .
The reason why the His
mutants have little activity could
be that the introduced side chains cannot stabilize the M2` site.
Indirect evidence for M2` destabilization in His
mutants
is shown by the weaker binding at M2, measured kinetically (34) and observed crystallographically. Further evidence is
provided by the ability of xylitol to eject the M2 when it is soaked
into the mutant enzyme.
In contrast to the A. missouriensis His-Asn/xylose structure in which M2 is absent and
acyclic-extended D-xylose is observed(19) , M2 and
-D-xylopyranose are observed in the structures of
His
-Ser (this study) and His
-Glu (this
study). The difference in M2 occupancy might be due to different metals
used in the two studies; they employed Mg
, while in
this study Mn
was used. Depending upon the
substitution, there is a 48-200-fold decrease in metal affinity
with Mg
compared to Mn
in S.
rubiginosus His
mutants(34) . The reason(s)
that different sugar conformations are observed might be because
different metals were used (as above) and/or that different
substitutions might affect substrate binding or catalysis differently.
It is unlikely these mutants operate using a different reaction
mechanism. Attempts to recover second-site mutations that restore
partial activity in either the E. coli or S. rubiginosus enzymes have yielded only reversions back to the original amino
acid residue (i.e. His
-Ser back to
His
; data not shown). Not all mutations at His
result in the same activity. His
-Gln has 3.4%
activity, His
-Ser, His
-Asn, and
His
-Glu have 0.5-0.8% activity, and
His
-Lys has no activity(30, 34) .
His appears to be important for maintaining the
structure around M2 when the substrate is extended. Both the structure
of His
-Ser/xylitol determined in this study and the
structure of His
-Asn/xylose from A. missouriensis(19) display similar sugar binding, show that M2 is
absent, and reveal new positions for metal ligands and other residues
which are located in and near the active site. These differences are
presumably caused directly and indirectly by bringing O1 and O2 of the
extended sugar near a M2 site with lower affinity for metal.
The functions of active site residues in D-xylose
isomerase was investigated kinetically and structurally. Some of the
main conclusions and supporting observations are summarized here. 1)
His is not essential for catalysis, but appears to be
responsible for anomeric recognition and to contribute to the
stabilization of the transition state via hydrogen bonding to O5.
His
may act as a base-catalyst but not likely as an acid
catalyst in ring-opening where enzyme activity is maximal. 2)
Phe
is not important for anomeric recognition or essential
for enzyme activity, but it clearly contributes to the optimal binding
of the transition state. 3) Lys
is important structurally
and probably catalytically. Lys
-Met has structural
perturbations at Glu
, Pro
,
Trp
, and Phe
and no activity. Because
residues located at the catalytic center only experience minor
perturbations, we suggest that Lys
has a direct role in
sugar extension and/or isomerization through interactions with O1 of
substrate. 4) Additional, unaccounted for electron density is observed
near M2 in the structure of His
-Ser/xylose and appears to
represent an alternate position for M2 (M2`). Comparing the peak
heights of M2 and M2` in the F
- F
map of the wild-type enzyme yields a ratio
M2:M2` of 13:1. Metal at the M2` position could be a major contributor
to catalysis coordinating both O1 and O2 of the acyclic-extended sugar.
The low activity of His
-Ser may be due to the inability
of Ser
to stabilize metal binding at the M2` position. 5)
His
is likely important because it coordinates metal at
both the M2 and M2` positions, as we suggest it is necessary for
catalysis. His
-Ser and His
-Glu mutants have
little activity and their structures have M1, M2, and
-D-xylopyranose bound to the active site. Xylitol binds
in an extended form to His
-Ser, but the metal at the M2
position is lost from the enzyme rather than simply being shifted.