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

The structure of the Mn2+-bound enzyme indicated that the catalytic site interconverts between two forms with the displacement of the metal ion to recognize both pyranose and furanose ri

L-rhamnose isomerase deduced from X-ray structures

Hiromi Yoshida1, Masatsugu Yamaji1,2, Tomohiko Ishii2, Ken Izumori3and Shigehiro Kamitori1

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: kamitori@med.kagawa-u.ac.jp

(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 Mn2+-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 Mn2+-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.

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 Mn2+, Cu2+, Co2+and Zn2+[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 Zn2+, as the atomic absorption spectrum of the purified enzyme showed the presence of Zn2+,

Mn2+ and Ni2+ in a ratio of 4 : 1 : 1 [7] The struc-ture with a specific metal ion, Mn2+, 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 Mn2+-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

Mn 2+

Mn 2+

H O

Base Base

L -rhamnose L -rhamnulose

1

2

3

4

5

6

1

2

3

4

5

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

CH3

H OH

HO H

HO H

CH3

O

CH2OH

O CH2 OH OH OH

OH

CH3 O

OH

HO

HO

H3C

OH

O

OH

OH

OH

HO

OH

H O

H OH

H OH

H OH

H OH

CH2OH

CH2OH

H OH

H OH

H OH

CH2OH

O

O

CH2OH

OH OH OH

CH 2 OH

“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).

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 (Mn2+) 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 Mn2+-bound form

As Zn2+, Mn2+ and Ni2+ 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 Mn2+-, Cu2+-, Co2+- and Zn2+-bound forms could be obtained

In all the enzymes, two metal ions bind to each com-ponent of the tetramer The Cu2+-, Co2+- and Zn2+ -bound forms have the same metal-coordinated struc-ture in all four molecules (Mol-A, Mol-B, Mol-C and Mol-D); however, the Mn2+-bound form has two metal-coordinated structures, as shown in Fig 3 The final electron density maps for Mn2+ ions in the two metal-coordinated structures are given in Fig S1 (see Supporting information) In Mol-A and⁄ or Mol-D, the structural Mn2+(Mn1) is coordinated by six

His281(ND), Asp327(OD) and two water molecules (W1 and W2), and the catalytic Mn2+ (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 W6 W6

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 Mn2+ions and red water

molecules The BC-form (Mol-B) is indicated

by green carbon amino acid residues, gray

Mn2+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.

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 Cu2+-,

Co2+- and Zn2+-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

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 hydrohydro-gen

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

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.

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

His101 Trp57

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 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.

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 1C4 and 4C1 chair conformations, respectively, because the C6 group should be equatorial (Fig 1A) Indeed, their crystal structures showed a-l-rhamnopyranose with a 1C4 chair conformation [22] and b-d-allopyranose with a

4C1 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 1C4 and⁄ or 4C1 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

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 pKa value of Asp291 is expected to be increased in the AD-form

N

N O

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

O O

Mn O H Mn

O

O OH OH

OH

O H H

O – –

Mn1 Mn2

Mn1 Mn2

Mn1 Mn2

Mn1 Mn2

Mn1 Mn2

Mn1

Mn2

Trp179

Asp291

Asp327

Asp291

Asp327

Asp291

Asp327

Asp327

Trp179

Asp327 Asp327

AD-form

AD-form BC-form

BC-form

–

– –

–

–

Asp291

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.

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

Completeness (%) 99.9 (99.9) 99.7 (99.2) 97.2 (96.5) 96.7 (96.8) 100.0 (99.9)

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)

Cell dimensions

Resolution (A ˚ ) 43.36–1.80 46.39–1.60 47.01–1.96 42.15–1.90 42.12–1.70

No of amino acids Mol-A 420 Mol-A 421 Mol-A 421 Mol-A 421 Mol-A 421

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 )

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 Mn2+-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 Cu2+-, Co2+

-and Zn2+-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 Mn2+

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 (Vmax 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 MnCl2, CuSO4, CoCl2 or ZnSO4 for 20 min at 20C Each sample was concentrated to

20 mgÆmL)1with 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