Báo cáo khoa học: Catalytic reaction mechanism of Pseudomonas stutzeri L-rhamnose isomerase deduced from X-ray structures doc

Catalytic reaction mechanism of Pseudomonas stutzeri
L-rhamnose isomerase deduced from X-ray structures
Hiromi Yoshida
1
, Masatsugu Yamaji
1,2
, Tomohiko Ishii
2
, Ken Izumori
3
and Shigehiro Kamitori
1
1 Life Science Research Center and Faculty of Medicine, Kagawa University, Japan
2 Faculty of Technology, Kagawa University, Japan
3 Rare Sugar Research Center and Faculty of Agriculture, Kagawa University, Japan
Introduction
l-Rhamnose isomerase (l-RhI), which catalyzes the
reversible isomerization of l-rhamnose to l-rhamnu-
lose, has been found to be involved in the metabolism
of l-rhamnose in Escherichia coli (Fig. 1A) [1,2], and
the X-ray structure of E. coli l-RhI was determined
[3]. Pseudomonas stutzeri l-RhI, with a broad substrate
specificity compared with E. coli l-RhI, can catalyze
not only the isomerization of l-rhamnose, but also
that between d-allose and d-psicose (Fig. 1A) [4–6]. As
d-allose and d-psicose are ‘rare sugars’, existing in
small amounts in nature, P. stutzeri l-RhI is exploited
for industrial applications in rare sugar production.
We have reported the structures of P. stutzeri l-RhI in
complexes with substrates (l-rhamnose and d-allose),
revealing a unique catalytic site recognizing both

l-rhamnose and d-allose [7].
l-RhI has structural homology with d-xylose isom-
erase (d-XI), in spite of the low sequence identity (13–
17%) between them. Both have a large domain with a
(b ⁄ a)
8
barrel and an additional small domain
Keywords
catalytic mechanism; hydride-shift;
L-rhamnose isomerase; rare sugar;
X-ray structure
Correspondence
S. Kamitori, Life Science Research Center
and Faculty of Medicine, Kagawa University,
1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa
761-0793, Japan
Fax: +81 87 891 2421
Tel: +81 87 891 2421
E-mail:
(Received 27 October 2009, revised 7
December 2009, accepted 15 December
2009)
doi:10.1111/j.1742-4658.2009.07548.x
l-Rhamnose isomerase (l-RhI) catalyzes the reversible isomerization of
l-rhamnose to l-rhamnulose. Pseudomonas stutzeri l-RhI, with a broad
substrate specificity, can catalyze not only the isomerization of l-rhamnose,
but also that between d-allose and d-psicose. For the aldose–ketose isomer-
ization by l-RhI, a metal-mediated hydride-shift mechanism has been
proposed, but the catalytic mechanism is still not entirely understood. To
elucidate the entire reaction mechanism, the X-ray structures of P. stutzeri

l-RhI in an Mn
2+
-bound form, and of two inactive mutant forms of
P. stutzeri l-RhI (S329K and D327N) in a complex with substrate ⁄ product,
were determined. The structure of the Mn
2+
-bound enzyme indicated that
the catalytic site interconverts between two forms with the displacement of
the metal ion to recognize both pyranose and furanose ring substrates.
Solving the structures of S329K–substrates allowed us to examine the
metal-mediated hydride-shift mechanism of l-RhI in detail. The structural
analysis of D327N–substrates and additional modeling revealed Asp327 to
be responsible for the ring opening of furanose, and a water molecule coor-
dinating with the metal ion to be involved in the ring opening of pyranose.
Structured digital abstract
l
MINT-7384817: L-RhI (uniprotkb:Q75WH8) and L-RhI (uniprotkb:Q75WH8) bind (MI:0407)
by X-ray crystallography (
MI:0114)
Abbreviations
CSD, Cambridge Structure Database;
D-XI, D-xylose isomerase; L-RhI, L-rhamnose isomerase; PDB, Protein Data Bank.
FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS 1045
composed of a-helices, which form a homotetramer,
and each subunit has two adjacent metal ions at the
catalytic site: one a ‘structural metal ion’ to aid sub-
strate binding, and the other a ‘catalytic metal ion’ to
help with the catalytic reaction [8]. Many structural
studies of d-XI have been performed to understand its
catalytic reaction mechanism [8–19]. Two types of

mechanism, the ene-diol mechanism [9] and the
hydride-shift mechanism [8,11–13,16], have been pro-
posed for the aldose–ketose isomerization of d-XI
based on the X-ray structures. According to the ene-
diol mechanism, two bases transfer a proton from O2
to O1, and a proton from C1 to C2, respectively, pro-
ducing ketose from aldose, as shown in Fig. 1B. Dur-
ing the reaction, the ene-diol intermediate is stabilized
by the metal ion. In the structures of d-XIs, a water
molecule coordinating to the metal ion was thought to
act as a base to transfer a proton from O2 to O1, but
a suitable base to transfer a proton from C1 to C2 has
been never found. It was also reported that protons of
C1 and ⁄ or C2 do not exchange with solvent [20]. Thus,
the hydride-shift mechanism was proposed and gener-
ally accepted, in which a hydride ion moves from C2
to C1, as shown in Fig. 1B. However, neutron-based
studies of d-XIs suggest that the possibility of the ene-
diol mechanism still remains [17,18].
The structure of P. stutzeri l-RhI without a suitable
base to transfer a proton between C2 and C1 seems to
support the metal-mediated hydride-shift mechanism
for aldose–ketose isomerization, but the catalytic reac-
tion mechanism is still not entirely understood. First,
the mechanism for the ring opening of a substrate is
unknown. In d-XI, two adjacent residues (His53 and
Asp56) are proposed to be responsible for the opening,
but the pair is not conserved in l-RhI. This suggests
that l-RhI has a different ring opening mechanism
from d-XI. Second, as the enzymatic activity of

P. stutzeri l-RhI is strongly dependent on the metal
ion species, the activity ratio being 100 : 35 : 19 : 10
for Mn
2+
,Cu
2+
,Co
2+
and Zn
2+
[6], the relationship
between the species of metal ion and enzymatic activity
needs to be elucidated. In a previously reported struc-
ture of P. stutzeri l-RhI, the bound metal ions were
refined as Zn
2+
, as the atomic absorption spectrum of
the purified enzyme showed the presence of Zn
2+
,
Mn
2+
and Ni
2+
in a ratio of 4 : 1 : 1 [7]. The struc-
ture with a specific metal ion, Mn
2+
, would be
required.
To obtain new insights into the overall catalytic reac-

tion mechanism of P. stutzeri l-RhI, we report here the
X-ray structures of an unliganded P. stutzeri l-RhI in
the Mn
2+
-bound form, and two inactive mutant forms
of P. stutzeri l-RhI, with substitutions of Ser329 with
Lys (S329K) and Asp327 with Asn (D327N), in a com-
plex with a substrate ⁄ product. The complex structures
of D327N with d-psicose and l-rhamnulose are the
first in which the bound substrate⁄ product has a fura-
nose ring conformation in the sugar isomerase.
H
O
H O H
Mn
2+
Mn
2+
HO
H
O
H O H
Base
Base
L-rhamnose L-rhamnulose
D-allose D-psicose
1
2
3
4

5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
(β-L-rhamnulofuranose)(α-L-rhamnopyranose)
(α-
D-psicofuranose)
(β-
D-allopyranose)
H
O
H OH
H OH
HO H

HO H
CH
3
H OH
HO H
HO H
CH
3
O
CH
2
OH
O
CH
2
OH
OH
OH
OH
CH
3
O
OH
HO
HO
H
3
C
OH
O

OH
OH
OH
HO
OH
H
O
H OH
H OH
H OH
H OH
CH
2
OH
CH
2
OH
H OH
H OH
H OH
CH
2
OH
O
O
CH
2
OH
OH
OHOH

CH
2
OH
ene-diol mechanism Hydride-shift mechanism
“Catalytic”
“Structural”
A
B
Fig. 1. (A) Chemical reactions catalyzed by
P. stutzeri
L-RhI with potential substrates.
(B) Two types of proposed catalytic mecha-
nism for aldose–ketose isomerization, the
ene-diol mechanism (left) and the hydride-
shift mechanism (right).
Catalytic mechanism of
L-rhamnose isomerase H. Yoshida et al.
1046 FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS
Results and Discussion
Overall structure of P. stutzeri l-RhI
The overall structure of P. stutzeri l-RhI has been
reported previously [7]. Briefly, the monomeric struc-
ture of P. stutzeri l-RhI comprises a large domain
(Phe50–Val356) with a (b ⁄ a)
8
barrel fold, and an addi-
tional small domain (N-terminus–Lys49 and Asp357–
C-terminus), and two metal ions (Mn
2+
) bind to the

centre of the barrel to form the catalytic site. The
enzyme forms a tetramer comprising Mol-A, Mol-B,
Mol-C and Mol-D, with a noncrystallographic 222
symmetry, having four catalytic sites. The pair Mol-A
and Mol-B and ⁄ or Mol-C and Mol-D with a two-fold
symmetry forms the accessible surface for substrate
binding, as shown in Fig. 2. Phe66 in the loop region
between the first b-strand and a-helix of Mol-A
approaches the catalytic site of Mol-B to interact with
a substrate, whereas no amino acid residue of Mol-A
approaches the catalytic sites of Mol-C and Mol-D.
Structure of the enzyme in the Mn
2+
-bound form
As Zn
2+
,Mn
2+
and Ni
2+
were found to bind to the
purified enzyme from the atomic absorption spectrum
in a previously reported study [7], the entire removal
of metal ions from the purified enzyme should be
required to obtain the enzyme with a specific metal
ion-bound form. We successfully prepared the enzyme
in a metal-free form by 5 mm EDTA treatment, and
the removal of metal ions was confirmed by X-ray
analysis (Table S1, see Supporting information). By
incubating this metal-free form with each specific metal

ion, the Mn
2+
-, Cu
2+
-, Co
2+
- and Zn
2+
-bound forms
could be obtained.
In all the enzymes, two metal ions bind to each com-
ponent of the tetramer. The Cu
2+
-, Co
2+
- and Zn
2+
-
bound forms have the same metal-coordinated struc-
ture in all four molecules (Mol-A, Mol-B, Mol-C and
Mol-D); however, the Mn
2+
-bound form has two
metal-coordinated structures, as shown in Fig. 3. The
final electron density maps for Mn
2+
ions in the two
metal-coordinated structures are given in Fig. S1 (see
Supporting information). In Mol-A and ⁄ or Mol-D, the
structural Mn

2+
(Mn1) is coordinated by six coordina-
tion bonds from Glu219(OE), Asp254(OD),
His281(ND), Asp327(OD) and two water molecules
(W1 and W2), and the catalytic Mn
2+
(Mn2) is
Mol-D
Mol-A
Mol-B
Mol-C
Phe66 (Mol-B)
Phe66 (Mol-A)
Fig. 2. Overall tetrameric structure of P. stutzeri L-RhI. The four
molecules are colored in yellow (Mol-A), green (Mol-B), magenta
(Mol-C) and light blue (Mol-D). The dark-colored part of each mole-
cule represents the additional small domain. The small spheres indi-
cate metal ions. Phe66 and the loop regions between the first
b-strand and a-helix of Mol-A and Mol-B are indicated by a stick
model, and black, respectively.
Lys221
Asp289
Asp291
Asp327
Asp254
Glu219
His257
His281
W1
W2

W3
W4
W5
W7
Mn1
Mn2
Lys221
Asp289
Asp291
Asp327
Asp254
Glu219
His257
His281
W1
W2
W3
W4
W5
W7
Mn1
Mn2
W6W6
Fig. 3. Stereoview of the two forms of
metal-bound structure of P. stutzeri
L-RhI in
the Mn
2+
-bound form. The AD-form (Mol-A)
is indicated by yellow carbon amino acid

residues, black Mn
2+
ions and red water
molecules. The BC-form (Mol-B) is indicated
by green carbon amino acid residues, gray
Mn
2+
ions and pink water molecules.
Selected interactions among amino acid
residues, metal ions and water molecules
are indicated by black (Mol-A) and gray
(Mol-B) dotted lines.
H. Yoshida et al. Catalytic mechanism of
L-rhamnose isomerase
FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS 1047
coordinated by His257(NE), Asp289(OD1) and four
water molecules (W2, W3, W4 and W5). The distance
between Mn1 and Mn2 is 4.2 A
˚
, and a water molecule
of W2 bridges the metal ions. This metal-coordinated
structure is equivalent to those found in the Cu
2+
-,
Co
2+
- and Zn
2+
-bound forms (Fig. S2, see Supporting
information). This is denoted as the ‘AD-form’. A sub-

strate binds to the catalytic site in the AD-form, as
described later. In Mol-B and ⁄ or Mol-C, Mn1 is coor-
dinated in the same way as in Mol-A and Mol-D, but
Mn2 is coordinated by His257(NE), Asp289(OD1),
Asp289(OD2), Asp291(OD) and two water molecules
(W6, W7). The distance between Mn1 and Mn2 is
5.2 A
˚
, and the water molecules W2 and W6 bridge the
metal ions. This metal-coordinated structure is denoted
as the ‘BC-form’.
A disordered catalytic metal ion was identified by
the high-resolution X-ray structure of Strepto-
myces olivochromogenes d-XI, showing that the dis-
placement of metal ions was involved in the catalytic
reaction [16]. Thus, it is likely that the positions of the
catalytic metal ions of P. stutzeri l-RhI also vary
between the AD- and BC-forms. Through metal-coor-
dinated structural change from the BC- to AD-form,
Mn2 moves by 1.90 A
˚
towards the substrate-accessible
surface, accompanied by the movement of W7 to the
position of W5, W6 to W3 and W3 to the solvent
channel. Mn2 in the AD-form attracts W2, leading to
the movement of Mn1, His281 and W1 by 0.65 A
˚
to
Mn2. The distance between Mn1 and Mn2 changes
from 5.2 A

˚
(BC-form) to 4.2 A
˚
(AD-form). Tempera-
ture factors of Mn2 (30.3, 26.9, 30.0 and 30.1 A
˚
2
for
Mol-A, Mol-B, Mol-C and Mol-D, respectively) are
significantly higher than those of Mn1 (14.6, 17.7, 22.2
and 15.5 A
˚
2
), supporting the high mobility of Mn2 in
the enzyme. The displacement of Mn2 does not affect
greatly the overall structure of the subunit. The small
movement of His281 causes side-chain conformational
changes of neighboring Phe280 and Leu255, but no
other significant structural differences between subunits
of the AD- and BC-forms were found. It is unclear
why the AD-form is found in Mol-A ⁄ Mo-D and the
BC-form in Mol-B ⁄ Mol-C.
Complex structure of S329K with the linear
conformation substrate
In previously reported structures of P. stutzeri l-RhI
in complexes with l-rhamnose and d-allose, there was
some ambiguity in the electron density of the bound
substrate, and it was difficult to discuss the precise
conformation of the substrate [7]. These X-ray struc-
tures and the structural comparison with Actinopla-

nes missouriensis d-XI complexed with d-sorbose [19]
showed that the substitution of Ser329 with Lys is
effective in increasing the attractive interactions
between a substrate and the enzyme without any spa-
tial change of the other amino acid residues at the
catalytic site, because A. missouriensis d-XI has inher-
ently Lys as a corresponding residue to Ser329,
directing its side-chain group to the substrate. We
prepared a mutant form through the substitution of
Ser329 with Lys (S329K), and successfully determined
the structure of its complexes, S329K–d-psicose
(ketose) and S329K–l-rhamnose (aldose). As expected,
the substituted Lys forms a hydrogen bond with
the substrate, stabilizing the complex. The enzymatic
activity of S329K is 2% of that of the wild-type
enzyme.
The catalytic site structure of S329K–d-psicose is
shown in Fig. 4A, with the electron density of the
bound d-psicose. Clear electron density gave the pre-
cise conformation of d-psicose, as indicated in Table 1.
O1, O2 and O3 of d-psicose strongly coordinate with
Mn1 and Mn2 with distances of 2.0–2.3 A
˚
, instead of
W3, W2 and W1 in the AD-form (Figs 3, 4A). As a
result of the strong metal coordination, two virtual
five-membered rings of O1, C1, C2, O2 and Mn2, and
of O2, C2, C3, O3 and Mn1, adopt an almost planar
structure within 0.03 A
˚

and 0.1 A
˚
, respectively.
Lys221, Asp327 and Glu219 form a hydrogen bond
with O1, O2 and O3, respectively, helping to fix the
substrate in the appropriate conformation for the cata-
lytic reaction. There are still two water molecules (W4
and W5) coordinating with Mn2, and they too form
hydrogen bonds with Asp291. W4 is thought to be a
catalytic water molecule responsible for the proton
transfer between O1 and O2, because it possibly forms
hydrogen bonds with O1 and O2 of the substrate. On
the opposite side to W4 [re-face side of the carbonyl
carbon (C2)], there is no base to transfer a proton
between C1 and C2, but a space along the C1–C2
bond surrounded by Trp179, Lys221 and His257. This
space is favorable for the hydride-shift between C1
and C2, because C1 and C2 are shielded from solvent
access, to prevent a water molecule as a nucleophile
attacking the carbonyl carbon. The indole ring of
Trp179 makes CH–p interaction with H1A on the
re-face side, and this may help the formation of a sta-
ble hydride ion (H
)
). Therefore, the presented X-ray
structure most probably supports the hydride-shift
mechanism for the isomerization reaction of P. stutzeri
l-RhI. As O5 forms a van der Waals’ contact with C2
on the si-face side of C2, H1B cannot shift to C2,
showing that l-RhI can strictly produce an aldose with

a right-hand configuration at the 2-position in
Catalytic mechanism of L-rhamnose isomerase H. Yoshida et al.
1048 FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS
Fischer’s projection through isomerization from the
ketose to aldose.
His101 forms a hydrogen bond with O4, and
Asp327 with O5, to recognize the hydroxyl groups at
the 4- and 5-positions of d-psicose. Trp57 exhibits
hydrophobic interaction with C6 of the substrate, but
O6 does not form a hydrogen bond with any amino
acid residue. This is because the inherent substrate of
l-rhamnose is a deoxy-sugar without a hydroxyl group
at the 6-position. The substituted Lys329 forms hydro-
gen bonds with O5, Asp327 and W4. The hydrogen
bond between Lys329 and the substrate may freeze the
conformation of a substrate to stabilize the enzyme–
substrate complex. The hydrogen bond between the
amino group of Lys329 and W4 possibly compensates
for the negative charge of W4 as a hydroxyl ion, lead-
ing to the inactivation of W4 as a catalytic water mole-
cule. This may be why the enzymatic activity of S329K
is 2% that of the wild-type enzyme.
The catalytic site structure of S329K–l-rhamnose,
with the electron density of the bound l-rhamnose, is
shown in Fig. 4B. Owing to H2, the virtual five-mem-
bered ring of O1, C1, C2, O2 and Mn2, and ⁄ or of O2,
C2, C3, O3 and Mn1, does not form a planar
Table 1. Torsion angles (deg) of the bound substrate ⁄ product.
O1–C1–C2–C3 C1–C2–C3–C4 C2–C3–C4–C5 C3–C4–C5–C6 C4–C5–C6–O6
D-Psicose )173 55 70 )173 64

L-Rhamnose )159 43 168 )177 )
D-Psicofuranose )173 96 29 )144 )154
L-Rhamnulofuranose )178 122 )18 146 –
His257
Lys221
Trp179
His101
Trp57
Asp291
Asp327
Glu219
Ser→Lys329
W4
W5
Mn2
Mn1
H1A
H1B
O1
O2
O3
O4
O5
O6
His257
Lys221
Trp179
His101
Trp57
Asp291

Asp327
Glu219
Ser→Lys329
W4
W5
Mn2
Mn1
H1A
H1B
O1
O2
O3
O4
O5
O6
His257
A
B
Lys221
Trp179
His101
Trp57
Asp291
Asp327
Glu219
Ser→Lys329
W4
W5
Mn2
Mn1

H2
O1
O2
O3
O4
O5
His257
Lys221
Trp179
His101
Trp57
Asp291
Asp327
Glu219
Ser→Lys329
W4
W5
Mn2
Mn1
H2
O1
O2
O3
O4
O5
Fig. 4. Stereoview of the linear conforma-
tion substrate-binding structure of S329K:
(A)
D-psicose (orange carbon) and (B)
L-rhamnose (blue carbon) with a simulated

annealing omit map at the 4.0r contour
level. Selected interactions among amino
acid residues, substrates, metal ions and
water molecules are indicated by dotted
lines. Hydrogen atoms involved in a hydride-
shift ride on C1 of
D-psicose and C2 of
L-rhamnose were identified by geometrical
calculations.
H. Yoshida et al. Catalytic mechanism of
L-rhamnose isomerase
FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS 1049
structure, and the distances from O1, O2 and O3 to
Mn1 and Mn2 are relatively long (2.3–2.6 A
˚
) compared
with those found in S329K–d-psicose. However, the
interactions between O1, O2 and O3 of l-rhamnose and
the enzyme, including metal ions, are almost identical to
those found in the bound d-psicose. H2 is located
between Trp179 and His257. As there is a space on the
re-face side of the carbonyl carbon (C1) surrounded by
Trp179, Lys221 and His257, H2 can easily attack C1
from the re-face side on a hydride-shift.
The torsion angle around the C3–C4 bond of the
bound substrate differs between l-rhamnose and d-psi-
cose (Table 1), because the 4- and 5-positions in
l-rhamnose have the opposite configuration to those
in d-psicose. The bound l-rhamnose forms hydrogen
bonds between O4 and Asp327, and O5 and His101,

whereas the bound d-psicose does so between O4 and
His101, and O5 and Asp327. This means that P. stut-
zeri l-RhI can recognize substrates with different con-
figurations of C4 and C5 by using His101 and Asp327,
and vice versa. The substituted Lys329 forms hydrogen
bonds with O4, Asp327 and W4, and Trp57 shows
hydrophobic interaction with the substrate, as found
in the complex with the bound d-psicose. Trp57 more
effectively recognizes the hydrophobic methyl group
(C6) of l-rhamnose.
Complex structure of D327 with the ring
conformation substrate
In the structure of S329K–d-psicose, O5 and C2 of
d-psicose form a van der Waals’ contact, and Asp327 is
located within hydrogen bond-forming distance of both
O2 and O5, suggesting that Asp327 acts as an acid–base
catalyst in the ring opening of d-psicose. We prepared a
mutant form with the substitution of Asp327 with Asn
(D327N), and tried to solve the X-ray structure of the
complex in which a substrate with a ring conformation
binds to the enzyme. As expected, no enzymatic activity
of D327N could be detected.
As shown in Fig. 5A, d-psicose with a ring confor-
mation was successfully found at the catalytic site of
Lys221
A
B
Trp179
His101Trp57
Asp291

Asp→Asn327
Glu219
W4
W5
Mn2
Mn1
O1
O2
O3
O4
O6
O5
Lys221
Trp179
His101Trp57
Asp291
Asp→Asn327
Glu219
W4
W5
Mn2
Mn1
O1
O2
O3
O4
O6
O5
Lys221
Trp179

His101
Trp57
Asp291
Asp→Asn327
Glu219
W4
W5
Mn2
Mn1
O1
O2
O3
O4
O5
Lys221
Trp179
His101
Trp57
Asp291
Asp→Asn327
Glu219
W4
W5
Mn2
Mn1
O1
O2
O3
O4
O5

Fig. 5. Stereoview of the ring conformation
substrate-binding structure of D327N: (A)
D-psicose (orange carbon) and (B) L-rhamnu-
lose (blue carbon) with a simulated anneal-
ing omit map at the 4.0r contour level.
Selected interactions among amino acid
residues, substrate, metal ions and water
molecules are indicated by dotted lines.
Catalytic mechanism of
L-rhamnose isomerase H. Yoshida et al.
1050 FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS
D327N, and its precise conformation is indicated in
Table 1. The bound d-psicose adopts a five-membered
ring structure with a-anomer (a-d-psicofuranose), hav-
ing a half-chair conformation; C2, C3, C5 and O5
form a plane within 0.08 A
˚
, and C4 deviates by 0.47 A
˚
from the plane. O1, O2 and O3 coordinate with Mn1
and Mn2 at distances of 2.0–2.6 A
˚
. Lys221, Glu219
and His101 form hydrogen bonds with O1, O3 and
O4, respectively. O6 does not form a hydrogen bond
with an amino acid residue, as found in S329K–d-psi-
cose. a -d-Psicofuranose is sandwiched between Trp57
and Trp179, and the indole ring of Trp179 forms a
nicely stacking interaction with a furanose ring.
It is difficult to identify the NE and OE atoms of

the substituted Asn327 at the present resolution. The
torsion angle around the CB–CG bond of 43° in
Asn327 is significantly different from that of 8° found
in Asp327 of the wild-type enzyme, and the coordina-
tion distance to Mn1 becomes 2.6 A
˚
from 2.2 A
˚
.If
OE of Asn327 coordinates with Mn1, the lone pair
electrons of OE are not directed to Mn1, but, if NE
does, it can direct its lone pair electrons to Mn1. In
addition, the opposite atom to the metal coordination
of Asn327 forms a hydrogen bond with a secondary
amino group of Trp57. Thus, we determined the posi-
tions of NE and OE atoms, as shown in Fig. 5A. The
amino group (NE) of Asn327 forms hydrogen bonds
possibly with O2 and O5 of a substrate to prevent ring
opening of the substrate and to help stabilize the
enzyme–substrate complex. Moreover, Asp327 at its
original position is expected to be located within
hydrogen bond-forming distance of O2 and O5, acting
as an acid–base catalyst for ring opening of a sub-
strate.
To elucidate the six-membered ring (pyranose ring)
structure of l-rhamnose, we also carried out X-ray
structure determination of D327N–l-rhamnose. How-
ever, unexpectedly, a product, l-rhamnulose with a
five-membered ring structure (b-l-rhamnulofuranose),
was found at the catalytic site of D327N, as shown

in Fig. 5B. This means that D327N can achieve the
ring opening of l-rhamnose followed by the isomeri-
zation of aldose to ketose. After the production of
l-rhamnulose, the O5 nucleophile attacks C2 (car-
bonyl carbon) to form a hemiacetal, b-l-rhamnulof-
uranose. As the enzymatic activity of D327N
towards l-rhamnose could not be detected with a
cystein–carbazole assay measuring the amount of
ketose produced [5,21], hydrogen bonds formed by
Asn327 could allow a product to anchor at the cata-
lytic site.
The bound b-l-rhamnulofuranose adopts a half-
chair conformation, but the ring conformation is dif-
ferent from that of the bound a-d-psicofuranose, as
shown in Fig. 5B and Table 1. In b-l-rhamnulofura-
nose, C2, C3, C4 and O5 form a plane within 0.11 A
˚
,
and C5 deviates by 0.30 A
˚
from the plane. Owing to
this ring puckering, b-l
-rhamnulofuranose shows
almost identical interaction with the enzyme as the
bound a-d-psicofuranose, in spite of the different con-
figurations of C4 and C5.
l-rhamnose and d-allose are expected to adopt the
six-membered ring structures with
1
C

4
and
4
C
1
chair
conformations, respectively, because the C6 group
should be equatorial (Fig. 1A). Indeed, their crystal
structures showed a-l-rhamnopyranose with a
1
C
4
chair conformation [22] and b-d-allopyranose with a
4
C
1
chair conformation [23]. To elucidate how the
enzyme recognizes a variety of pyranose ring confor-
mations, the modeling of plausible pyranose-bound
structures was performed by the least-squares fitting of
O1, O2, O3, O4 and O5 between the bound a-d-psicof-
uranose and b- d-allopyranose, and between the bound
b-l-rhamnulofuranose and a-l-rhamnopyranose, as
shown in Fig. 6.
In both models, the bound substrate is located in
the hydrophobic pocket formed by Trp57, Phe131,
Trp179 and Phe66* from Mol-B without any steric
hindrance from amino acid residues of the enzyme.
Trp179 nicely stacks with a pyranose ring. O2 and O3
coordinate with Mn1, but O1 is unlikely to coordinate

with Mn2 in the AD-form because O1 of b-d-allopyra-
nose and ⁄ or C1 of a-l-rhamnopyranose is too close to
Mn2. Thus, a substrate with a pyranose ring is thought
to bind to the catalytic site in the BC-form, although
O1 of the substrate is not within coordination distance
of Mn2 in the BC-form. Asp327 cannot form a hydro-
gen bond with O1 and O5 of the substrate, suggesting
that it is not involved in the opening of a pyranose
ring. As W4 possibly forms hydrogen bonds with O1
and O5 of both substrates, it may act as an acid–base
catalyst in ring opening. This is supported by the find-
ing that the complex structure of D327N–l-rhamnu-
lose (furanose) was obtained by incubating D327N
with l-rhamnose (pyranose).
The hydrophobic pocket recognizing the sugar seems
to be able to accept various sugar ring conformations,
for example pyranose and ⁄ or furanose ring, the
1
C
4
and ⁄ or
4
C
1
chair conformation, and a- and ⁄ or b-ano-
mer. For isomerization to occur, a substrate first needs
to coordinate Mn1 with O2 and O3 on the same side
of the sugar ring, and the enzyme may strictly recog-
nize the configuration of the 2- and 3-positions at this
stage. However, other anomers of furanoses, b-d-psi-

cofuranose and a-l-rhamnulofuranose, in which O2
and O3 are on the opposite side of the sugar ring to
H. Yoshida et al. Catalytic mechanism of L-rhamnose isomerase
FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS 1051
each other, seem to be poorly recognized as a substrate
by the enzyme.
The catalytic reaction mechanism
From the results of the X-ray structural study, we
deduced the catalytic reaction mechanism of P. stutzeri
l-RhI, as shown in Fig. 7.
Before the binding of a substrate, the catalytic site is
expected to interconvert between two forms (AD- and
BC-forms) with the displacement of Mn2 (Fig. 7A, D).
In the BC-form, a catalytic water molecule (W4) is
activated to become a hydroxyl ion (OH
)
) by coordi-
nating with Mn2. Asp291 helps the activation of W4
by removing a proton, because the pK
a
value of
Asp291 is expected to be increased in the AD-form
NH
NH
O
H
H
H
O
H

O O
Mn
O
H
H
H
O
H
Mn
O
O
H
O
H
H
O
H
O OH
HO
–
Mn
H
O
H
Mn
O O
H
O
H
O OH

HO
–
Mn
O O
O
OH
O
OH
OH
Mn
H
H
O
H
O OH
OH
Mn
O O
O
OH
OH
Mn
OH
O
H
H
H
H
O
H

O OH
O
Mn
O O
O
OH
OH
Mn
OH
O
H
H
H
H
O
H
H
O O
Mn
O
H
Mn
O
O
OH
OH
OH
OH
H
O

–
–
Mn1
Mn2
Mn1
Mn2
Mn1
Mn2
Mn1
Mn2
Mn1
Mn2
Mn1
Mn2
Trp179
Asp291
Asp327
Asp291
Asp327
Asp291
Asp327
Asp291
Asp291
Asp327
Trp179
Asp327
Asp327
AD-form
AD-form
BC-formBC-form

–
–
–
–
–
–
–
–
Asp291
AD-form
AD-form
AB C
DE F
Fig. 7. The proposed catalytic reaction mechanism of P. stutzeri L-RhI. The catalytic water molecule (W4) is highlighted by a red ellipsoid.
Yellow and green circles represent metal ions in two metal coordination forms, AD- and BC-forms, respectively.
Asp289
Asp291
Asp327
Asp254
Glu219
His257
His281
Trp179
Phe131
His101
W4
Lys221
Phe66*
Mn1
Mn2

Trp57
Asp289
Asp291
Asp327
Asp254
Glu219
His257
His281
Trp179
Phe131
His101
W4
Lys221
Phe66*
Mn1
Mn2
Trp57
O1
O5
O4
O1
O5
O4
O6
O2
O2
O3
O6
O2
O2

O3
Fig. 6. Stereoview of the modeling structure of pyranose ring conformation substrates, D-allose (orange carbon) and L-rhamnose (blue car-
bon), binding to the catalytic site of P. stutzeri
L-RhI (Mol-A), with Mn
2+
(black). Phe66* shown by green carbons belongs to Mol-B. Two
Mn
2+
ions in the BC-form are also superimposed by gray spheres.
Catalytic mechanism of
L-rhamnose isomerase H. Yoshida et al.
1052 FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS
compared with the BC-form, where Asp291 coordi-
nates directly with Mn2 to stabilize its ionization state.
From the presented X-ray structures, it is difficult to
determine whether or not W4 is a hydroxyl ion (OH
)
).
However, high-resolution X-ray crystallography and
neutron diffraction studies of d-XI clearly show that
the catalytic water molecule is activated as a hydroxyl
ion (OH
)
) concurrently with the displacement of a cat-
alytic metal ion [16]. It is probable that W4 is acti-
vated through the metal-coordinated structural change
from the BC- to the AD-form in l-RhI. A ketose with
a furanose ring binds to the AD-form (Fig. 7B), and
O1, O2 and O3 coordinate with Mn1 and Mn2.
Asp327 acts as an acid–base catalyst in ring opening,

helping to transfer a proton from O2 to O5. After the
ring has been opened, the catalytic water molecule
(W4) mediates the transfer of a proton from O1 to O2,
and a hydride (H1A), shielded by Trp179 from solvent
access, attacks C2, producing an aldose with a hydro-
xyl group (O2) having a right-handed configuration in
Fischer’s projection (Fig. 7C). An aldose with a pyra-
nose ring probably binds to the BC-form (Fig. 7E),
because O1 seems to be too close to Mn2 in the AD-
form, supposing that O2 and O3 coordinate with Mn1,
Table 2. X-ray data collection and refinement statistics. R
merge
= RR|I
i
) <I>| ⁄ R<I>. Values in parentheses are of the high-resolution bin
(1.86–1.80 A
˚
for wild-type–Mn, 1.66–1.60 A
˚
for S329K–
D-psicose, 2.02–1.95 A
˚
for S329K–L-rhamnose, 1.97–1.90 A
˚
for D327N–D-psicose and
1.76–1.70 A
˚
for D327N–
L-rhamnulose).
Data set

Mn
2+
-bound
form S329K–
D-psicose S329K–L-rhamnose D327N–D-psicose D327N–L-rhamnulose
Beam line KEK PF KEK PF SPring-8 KEK PF KEK PF
BL-6A BL-17A BL-38B1 BL-6A BL-6A
Wavelength (A
˚
) 0.978 1.0000 1.0000 0.978 0.978
Temperature (K) 100 100 100 100 100
Resolution (A
˚
) 50–1.80 50–1.60 50–1.95 50–1.90 50–1.70
No. of measured refs. 560 757 766 234 370 304 441 658 698 734
No. of unique refs. 150 765 208 180 112 393 128 987 185 271
Completeness (%) 99.9 (99.9) 99.7 (99.2) 97.2 (96.5) 96.7 (96.8) 100.0 (99.9)
R
merge
0.065 (0.284) 0.045 (0.124) 0.061 (0.246) 0.083 (0.347) 0.064 (0.335)
I
o
⁄ r(I
o
) 11.0 (5.3) 19.9 (10.7) 11.0 (3.1) 8.8 (3.4) 11.5 (4.1)
Space group P2
1
P2
1
P2

1
P2
1
P2
1
Cell dimensions
A (A
˚
) 78.647 74.626 75.018 74.762 74.700
B (A
˚
) 105.115 104.643 104.842 104.732 104.634
C (A
˚
) 102.526 108.424 109.936 115.035 115.096
b (deg) 102.799 107.349 106.917 108.242 108.142
Resolution (A
˚
) 43.36–1.80 46.39–1.60 47.01–1.96 42.15–1.90 42.12–1.70
Completeness (%) 96.6 98.8 94.0 92.3 96.2
R
factor
0.165 0.163 0.183 0.176 0.165
R
free
0.197 0.184 0.225 0.211 0.188
Rmsd bond lengths (A
˚
) 0.005 0.004 0.005 0.005 0.005
Rmsd bond angles (deg) 1.2 1.2 1.1 1.1 1.2

No. of amino acids Mol-A 420 Mol-A 421 Mol-A 421 Mol-A 421 Mol-A 421
Mol-B 418 Mol-B 420 Mol-B 420 Mol-B 421 Mol-B 421
Mol-C 418 Mol-C 426 Mol-C 426 Mol-C 428 Mol-C 428
Mol-D 418 Mol-D 418 Mol-D 426 Mol-D 419 Mol-D 419
No. of solvent molecules 1770 2101 1259 1436 1899
No. of ligand Mn
2+
88 8 8 8
Sugar 0 4 4 4 4
Ramachandran plot
(%)favored ⁄ additional
90.9 ⁄ 8.5 90.9 ⁄ 8.5 90.9 ⁄ 8.4 90.5 ⁄ 8.9 91.1 ⁄ 8.4
B-factor (A
˚
2
)
Protein 13.1 10.8 20.7 14.5 14.7
Sugar 16.1 37.5 26.2 13.5
Mn1 (Structural metal ion) 17.5 13.6 25.3 18.4 11.4
Mn2 (Catalytic metal ion) 29.3 12.3 32.0 17.3 10.0
Solvent 22.3 21.8 24.8 20.8 24.8
PDB code 3ITX 3ITV 3ITT 3ITO 3ITL
H. Yoshida et al. Catalytic mechanism of
L-rhamnose isomerase
FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS 1053
as shown in Fig. 6. The catalytic water molecule (W4)
may act as an acid–base catalyst in ring opening, after
which Mn2 moves to a position in the AD-form to
activate W4, and mediates the transfer of a proton
from O2 to O1, to permit a hydride (H2) to attack C1,

giving a ketose (Fig. 7F).
Interconversion between the two forms (AD- and
BC-forms) with the displacement of Mn2 is very
important to the recognition of both pyranose and
furanose ring substrates. In the Mn
2+
-bound form, the
structural energy of the two forms is thought to be
almost equal, allowing easy interconversion between
them, because the two conformers were found equally
in the X-ray structure. However, for Cu
2+
-, Co
2+
-
and Zn
2+
-bound enzymes, the AD-form may be more
stable than the BC-form, and interconversion does not
occur easily. This may be why P. stutzeri l-RhI has
maximum enzymatic activity in the presence of Mn
2+
.
Based on the structure of S. olivochromogenes d-XI
complexed with a-d-glucopyranose, which is a suitable
substrate, as is d-xylose, a pair of adjacent residues
(His53 and Asp56) was proposed to be involved in ring
opening [16]. However, there is no corresponding pair
of His and Asp residues in P. stutzeri l-RhI, only a
His residue (His101). This may be a result of a differ-

ence in metal coordination with a ring conformation
between d-XI and l-RhI. The two hydroxyl groups
coordinating with Mn1 must be in the right-handed
configuration in Fischer’s projection, otherwise steric
hindrance occurs between the bound substrate and
Trp179 (Figs 4, 5). In d-XI, O2 and O4 in the right-
handed configuration coordinate with Mn1, and O1
and O2 coordinate with Mn2 when the hydride-shift
occurs. However, in the binding of a ring conforma-
tion, O3 and O4 coordinate with Mn1, because O2
and O4 in a ring conformation are too far from each
other to coordinate with Mn1. Consequently, O5 of a
ring conformation substrate is close to His53 (His101
in l-RhI). His53 attaches a proton to O5 and the
water molecule forming a hydrogen bond to Asp56
removes a proton from O1, which is followed by ring
opening. On changing to a linear conformation, the
metal coordination structure changes drastically so
that O2 (instead of O3) coordinates to Mn1, with O4.
However, in l-RhI, the bound substrate with a ring
conformation inherently coordinates to Mn1 with O2
and O3, and the metal coordination structure is not
changed as much by ring opening. O5 of the ring con-
formation is close to Asp327 (furanose) or W4 (pyra-
nose) not His101. Therefore, it could be that Asp327
and the catalytic water molecule (W4) are responsible
for the ring opening of furanose and pyranose, respec-
tively, in l-RhI.
Materials and methods
Site-directed mutagenesis and purification of the

enzyme
Mutant forms of P. stutzeri l-RhI were prepared using
recombinant E. coli JM 109 cells. Site-directed mutagenesis
was carried out using a plasmid, pOI-02, encoding the
l-RhI gene [6] and the Quick Change Kit (Stratagene, La
Jolla, CA, USA) for the construction of S329K and
D327N. The oligonucleotides used were: for S329K:
forward primer, 5¢-GATCGACCAGAAGCACAACGTC
AC-3¢; reverse primer, 5¢-GTGACGTTGTGCTTCTGGTC
GATC-3¢; for D327N: forward primer, 5¢-CCACATGAT
CAACCAGTCGC-3¢; reverse primer, 5¢-GCGACTGGTT
GATCATGTGG-3¢. The mutant forms were purified in the
same way as wild-type P. stutzeri l-RhI, as reported previ-
ously [7]. The enzymatic activity (V
max
for l-rhamnose as
a substrate) of the mutant enzymes was measured by a
cystein–carbazole assay, detecting the amount of ketose
(l-rhamnulose) produced, using the calorimetric method
[5,21].
Protein preparation for crystallization
The purified enzyme was dialyzed against a buffer solution
(5 mm Tris ⁄ HCl and 5 mm EDTA, pH 8.0) to remove
bound metal ions retained from the culture medium. After
the buffer had been replaced with 5 mm Hepes, pH 8.0, by
dialysis to remove EDTA, the enzyme solution was concen-
trated to 1–2 mgÆmL
)1
to prepare P. stutzeri l-RhI in
metal-free form. For the preparation of the enzyme

in metal-bound form, the enzyme solution was incubated
in the presence of 1 mm MnCl
2
, CuSO
4
, CoCl
2
or ZnSO
4
for 20 min at 20 °C. Each sample was concentrated to
20 mgÆmL
)1
with a Microcon YM-10 filter (Millipore, Bill-
erica, MA, USA). Crystals of P. stutzeri l-RhI in metal-free
and metal-bound forms, and mutant enzymes, were grown
by the vapor diffusion method using a protein solution
(20 mgÆmL
)1
) and a reservoir solution [7–8% (w ⁄ v) polyeth-
ylene glycol 20 000 and 50 mm Mes buffer (pH 6.3)].
Crystals of complexes with d-psicose and ⁄ or l-rhamnose
were obtained by a soaking method, and incubation for
24–31 h with an additional 0.5 lL of 100 mm substrate
solution.
Data collection and structural determination
Crystals were flash-cooled in liquid nitrogen at 100 K and
X-ray diffraction data were collected on the BL-6A and
BL-17A beam lines in the Photon Factory (Tsukuba,
Japan), and the BL38B1 beam line in SPring-8. Diffraction
data were processed using the programs hkl2000 [24] and

the ccp4 program suite [25]. Data collection statistics and
scaling results are listed in Table 2. The initial phases were
Catalytic mechanism of L-rhamnose isomerase H. Yoshida et al.
1054 FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS
determined by a molecular replacement method with the
program molrep [26] in the ccp4 program suite, using the
structure of wild-type P. stutzeri l-RhI [Protein Data Bank
(PDB) code 2HCV] as a probe model [7]. Further model
building was performed with the programs coot [27] in the
ccp4 program suite, and x-fit [28] in the xtalview
program system [29], and the structure was refined using
the programs refmac5 [30] and cns [31]. Water molecules
were gradually introduced if the peaks above 3.5r in the
(F
o
) F
c
) electron density map were in the range of a
hydrogen bond. In a Ramachandran plot [32], the number
of residues in most favored regions was determined by the
program procheck [33]. Refinement statistics are listed in
Table 2. Data collection and refinement statistics of the
enzymes in metal-free and Cu
2+
-, Co
2+
- and Zn
2+
-bound
forms, and their metal-binding structures, are given in

Table S1 with the PDB code. Figures 2–4 were illustrated
by the program pymol [34].
Modeling of plausible pyranose-bound structures
From the Cambridge Structure Database (CSD) [35], the
atomic coordinates of b-d-allopyranose (CSD record
COKBIN) and a-l-rhamonopyranose (CSD record RHA-
MAH12) were obtained. In the structure of b-d-allopyra-
nose, the torsion angle of C4–C5–C6–O6 was changed from
45° to 180°, because the bound a-d-psicofuranose has a tor-
sion angle of 154° (trans conformation) to avoid unusual
short contacts with amino acid residues. The structure was
further optimized by mopac2002 [36] in the Winmopac sys-
tem (Fujitsu Ltd., Japan) [37] using the AM1 Hamiltonian.
The optimized structures were added to the catalytic site of
the enzyme by the least-squares fitting of O1, O2, O3, O4
and O5 with the bound a-d-psicofuranose and ⁄ or b-l-
rhamnulofuranose in the enzyme using a locally developed
program, because the hydroxyl groups of a substrate are
expected to mainly interact with the enzyme. Figure 6 was
illustrated by the program pymol [33].
PDB accession numbers
The atomic coordinates and structure factors of P. stutzeri
l-RhI in Mn
2+
-bound form (PDB code 3ITX), S329K–
d-psicose (PDB code 3ITV), S329K–l-rhamnose (PDB code
3ITT), D327N–d-psicose (PDB code 3ITO) and D327N–
l-rhamnulose (PDB code 3ITL) have been deposited in the
Protein Data Bank, Research Collaboration for Structural
Bioinformatics, Rutgers University, New Brunswick, NJ,

USA.
Acknowledgements
This study was supported in part by the National Pro-
ject on Protein Structural and Functional Analyses,
the ‘intellectual-cluster’, and a Grant-in-Aid for Young
Scientist (B) (19770085) from the Ministry of Educa-
tion, Culture, Sports, Science and Technology of
Japan, and by the fund for Young Scientists 2007–8
and Characteristic Prior Research 2009 from Kagawa
University. This research was performed with the
approval of the Photon Factory Advisory Committee
and the National Laboratory for High Energy Physics,
and SPring-8, Japan.
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Catalytic mechanism of L-rhamnose isomerase H. Yoshida et al.

1056 FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS
Supporting information
The following supplementary material is available:
Fig. S1. The final 2F
o
) F
c
electron density for the
metal-binding sites of the Mn
2+
-bound form.
Fig. S2. Stereoview of the superimposition of the
enzymes in the Mn
2+
-bound form, metal-free form,
Cu
2+
-bound form, Co
2+
-bound form and Zn
2+
-
bound form.
Table S1. Data collection and refinement statistics of
the enzymes in the metal-free form, Cu
2+
-bound form,
Co
2+
-bound form and Zn

2+
-bound form.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
by the authors. Such materials are peer-reviewed and
may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
H. Yoshida et al. Catalytic mechanism of L-rhamnose isomerase
FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS 1057