Enzymatic characterization of 5-methylthioribose 1-phosphate isomerase from Bacillus subtilis

Article abstract of DOI:10.1271/bbb.70209

The product of the mtnA gene of Bacillus subtilis catalyzes the isomerization of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P). The catalysis of MtnA is a novel isomerization of an aldose phosphate harboring a phosphate group on the hemiacetal group. This enzyme is distributed widely among bacteria through higher eukaryotes. The isomerase reaction analyzed using the recombinant B. subtilis enzyme showed a Michaelis constant for MTR-1-P of 138μM, and showed that the maximum velocity of the reaction was 20.4μmol min^-1 (mg protein)^-1. The optimum reaction temperature and reaction pH were 35°C and 8.1. The activation energy of the reaction was calculated to be 68.7 kJ mol^-1. The enzyme, with a molecular mass of 76 kDa, was composed of two subunits. The equilibrium constant in the reversible isomerase reaction [MTRu-1-P]/[MTR-1-P] was 6. We discuss the possible reaction mechanism.

Full text of DOI:10.1271/bbb.70209

Biosci. Biotechnol. Biochem., 71 (8), 2021–2028, 2007
Enzymatic Characterization of 5-Methylthioribose 1-Phosphate
Isomerase from Bacillus subtilis
Yohtaro SAITO,1 Hiroki ASHIDA,1 Chojiro KOJIMA,1 Haruka TAMURA,2
y
1;
Hiroyoshi MATSUMURA,2 Yasushi KAI,2 and Akiho YOKOTA
1Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST),
8916-5 Takayama, Ikoma, Nara 630-0192, Japan
2Department of Materials Chemistry, Graduate School of Engineering, Osaka University,
2-1 Yamada-oka, Suita, Osaka 565-0871, Japan
Received April 12, 2007; Accepted May 15, 2007; Online Publication, August 7, 2007
[doi:10.1271/bbb.70209]
The product of the mtnA gene of Bacillus subtilis
catalyzes the isomerization of 5-methylthioribose 1-
phosphate (MTR-1-P) to 5-methylthioribulose 1-phos-
phate (MTRu-1-P). The catalysis of MtnA is a novel
isomerization of an aldose phosphate harboring a
phosphate group on the hemiacetal group. This enzyme
is distributed widely among bacteria through higher
eukaryotes. The isomerase reaction analyzed using the
recombinant B. subtilis enzyme showed a Michaelis
constant for MTR-1-P of 138 ꢀM, and showed that the
maximum velocity of the reaction was 20.4 ꢀmol minÀ1
(mg protein)À1. The optimum reaction temperature and
reaction pH were 35 ꢀC and 8.1. The activation energy
of the reaction was calculated to be 68.7 kJ molÀ1. The
enzyme, with a molecular mass of 76 kDa, was composed
of two subunits. The equilibrium constant in the
reversible isomerase reaction [MTRu-1-P]/[MTR-1-P]
was 6. We discuss the possible reaction mechanism.
The methionine salvage pathway (MSP) is a meta-
bolic pathway for recovery of the reduced sulfur in 5-
methylthioribose (MTR), which is formed in the syn-
thesis of polyamines, as methionine.2) The pathway is
distributed widely among bacteria, yeasts, plants, and
animals, and salvages the reduced sulfur into methionine
after the synthesis of polyamines and other compounds
such as mugineic acid and ethylene that are specific to
individual organisms.3) MSP has attracted particular
attention, as it is deleted in a variety of human tumor
tissues4) and the analogs of MTR are growth inhibitors
of the malarial protozoan Plasmodium falciparum.5) The
genes and the reactions of the whole MSP from MTR to
methionine were deduced initially in Klebsiella pneu-
moniae,6) and have been identified using Bacillus
subtilis,7) where the reaction from MTR-1-phos-
phate (MTR-1-P) to 5-methylthioribulose-1-phosphate
(MTRu-1-P) is catalyzed by MtnA or MTR-1-P isomer-
ase (Fig. 1).
Key words: aldose-ketose isomerase; 5-methylthiori-
bose-1-phosphate isomerase; eukaryotic ini-
tiation factor 2B ꢀ-like protein; methionine
salvage pathway; Bacillus subtilis
MtnA has 20% similarity in amino acid sequence to
that of the ꢀ-subunit of eukaryotic initiation factor (eIF)
2B, and was originally identified as an eIF2Bꢀ-like
protein (eIF2Bꢀ-LP) (Fig. 2A).8) eIF2B is composed of
five (ꢀ to ") subunits and is involved in the GTP/GDP
exchange of eIF2 in the eukaryotic translation step.9,10)
The GTP-binding form of eIF2 (eIF2-GTP) transfers
initiator methionyl-tRNA (Met-tRNAiMet) to 40S ribo-
somes as an eIF2-GTP/Met-tRNAiMet complex. GTP is
hydrolyzed to GDP, and then eIF2-GDP is released from
40S ribosomes. The GDP on eIF2 must be changed to
GTP by the GTP/GDP exchange reaction of eIF2B to
participate in the next translational initiation. eIF2Bꢀ
has been found to participate in the regulation of the
GTP/GDP exchange reaction of the eIF2B complex, but
The major forms of sulfur and nitrogen occur in
nature as oxidized inorganic compounds. Both elements
need 8 electrons per molecule to be reduced to the
sulfhydryl group and ammonia, respectively. Since the
conversion of CO2 to glucose uses 4 electrons per
molecule of CO2, the sulfhydryl and amino groups in
living entities are high energy-carrying groups.1) Once
sulfur and nitrogen are reduced, organisms usually
reutilize the reduced forms many times without oxida-
tion.
y
To whom correspondence should be addressed. Tel: +81-743-72-5560; Fax: +81-743-72-5569; E-mail: yokota@bs.naist.jp
Abbreviations: MSP, methionine salvage pathway; MTR, 5-methylthioribose; MTR-1-P, 5-methylthioribose 1-phosphate; MTRu-1-P, 5-
methylthioribulose 1-phosphate; eIF, eukaryotic initiation factor; eIF2Bꢀ-LP, eukaryotic initiation factor 2B ꢀ-like protein; Met-tRNAiMet, initiator
methionyl-tRNA; HK-MTPenyl-1-P, 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate; NMR, nuclear magnetic resonance; R5P, ribose 5-
phosphate; PDB, Protein Data Bank (http://www.rcsb.org/pdb/)

2022
Y. SAITO et al.
2-
50 mM Tris–HCl buffer (pH 8.0) containing 0.1 M NaCl.
OPO3
H
CH3SH2C5
OPO3
The column was developed with a fast protein liquid
chromatography system (Amersham Pharmacia). The
dye-binding method of Bradford12) was used to deter-
mine protein concentration. The purity of the prepared
recombinant enzyme was checked on SDS–polyacryl-
amide gel electrophoresis gel by staining with Coomas-
sie Brilliant Blue R-250.13)
2-
H
1
O
O
4
1
2
3
4
5
H
H
H
H
H
OH
OH
H
H
H
3
2
HO
OH
SCH3
MTRu-1-P
MTR-1-P
Preparation of MTR-1-P and quantification of MTR-
1-P and MTRu-1-P. MTR-1-P was synthesized from S-
adenosylmethionine through hydrolysis of the adenine
base by HCl, and C1 of the ribose moiety was phos-
phorylated by ATP with MtnK,7) an MTR kinase.14)
MTR-1-P was desalted using a column (2:6 ꢁ 65 cm) of
Sephadex G-10 (Amersham Pharmacia).
Fig. 1. Reaction Scheme of MTR-1-P Isomerase.
MTR-1-P isomerase, MtnA, catalyzes the reaction for isomer-
ization of MTR-1-P to MTRu-1-P. Numbers near carbons stand for
the carbon numbers of the sugar phosphates.
MTR-1-P and MTRu-1-P in the reaction mixture were
applied to a CarboPac PA1 column (Dionex, Osaka,
Japan) equilibrated with 100 mM NaOH. The column
was developed using a linear gradient of 0 to 0.5 M
potassium acetate in 100 mM NaOH. The sugar phos-
phates thus separated were detected with a pulsed
amperometry system and quantified as to phosphate
content using a phosphate assay kit (Phosphor C, Wako,
Osaka, Japan). The flow rate (1 ml minꢂ1) and elution
gradients were controlled with a gradient pump GP40
(Dionex).
its protein structure and exact function in the complex
remain unclear. In this context, it is intriguing that
eIF2Bꢀ-related MtnA is an MTR-1-P isomerase func-
tioning in MSP.
After our identification of the MtnA function, E2B2,
the eIF2Ba-LP in Thermococcus kodakaraensis, was
identified as ribose 1,5-bisphosphate (RBP) isomerase
(Fig. 2).11) This enzyme supplies form III ribulose 1,5-
bisphosphate carboxylase/oxygenase with ribulose 1,5-
bisphosphate for CO2 fixation. It also catalyses the
isomerization reaction of a cyclic sugar harboring a
phosphate group at C1, as does MtnA of B. subtilis, but
there have been no previous reports of the isomerase
reaction of cyclic sugars with the phosphate group on
the C1 aldehyde carbon.
MTR-1-P has the phosphate group on C1, which
consequently is not able to give rise to a free aldehyde
structure. Thus its isomerase reaction should be different
from those of ordinary sugars that have no modification
on the C1 aldehyde carbon. We were interested in the
reaction mechanism of this new enzyme MTR-1-P
isomerase, and in this study analyzed the general
enzymatic properties of the protein. We propose a
mechanism for the isomerase reaction of this enzyme.
Assay of MTR-1-P isomerase. MTR-1-P isomerase
was assayed in a coupling reaction with MtnB and
MtnW, previously identified as MTRu-1-P dehydratase
and 2,3-diketo-5-methylthiopentyl-1-phosphate enolase
respectively.2,7,15) In this assay, the reaction product
MTRu-1-P is converted to 2-hydroxy-3-keto-5-methyl-
thiopentenyl-1-phosphate (HK-MTPenyl-1-P) by se-
quential reactions of MtnB and MtnW. The reaction
was started by adding 2 mM MTR-1-P to a mixture
containing 50 mM Tris–HCl buffer (pH 8.1), 1 mM
MgCl2, 0.5 mg of MTR-1-P isomerase, 5 mg of MtnB,
and 15 mg of MtnW. The final reaction volume was 1 ml
and the reaction temperature 35 ꢀC, unless otherwise
1
stated. H-nuclear magnetic resonance (NMR) analysis
Materials and Methods
of the reactions from MTR-1-P to HK-MTPenyl-1-P7)
indicated that virtually all of the MTR-1-P added to the
reaction mixture was converted into HK-MTPenyl-1-P.
This suggests that the chain of reactions from MtnA to
MtnW is strongly inclined toward the formation of HK-
MTPenyl-1-P; thus, from the light absorbance of HK-
MTPenyl-1-P formed from a known concentration of
MTR-1-P, the molecular extinction coefficient of HK-
Preparation of MTR-1-P isomerase. MTR-1-P iso-
merase was expressed in Escherichia coli as (His)6-
tagged recombinant protein and purified using Ni-NTA
His-Bind resin (Novagen, Madison, WI), as described
previously.7) After purification by the His-tag affinity
system, MTR-1-P isomerase fraction was passed
through a PD-10 column (Amersham Pharmacia, Up-
psala, Sweden) equilibrated with 50 mM Tris–HCl buffer
(pH 8.0) containing 150 mM NaCl and 2.5 mM CaCl2,
and the protein fractions were collected. The (His)6-tag
was excised with 1.3 mg thrombin per mg of MTR-1-P
isomerase for 12 h at 22 ꢀC. MTR-1-P isomerase was
further purified on a Hiload Sephadex 200 16/60
column (Amersham Pharmacia) equilibrated with
MTPenyl-1-P at 280 nm was determined to be 9,500
15)
M
ꢂ1 cmꢂ1
.
The responses of the activity to temper-
ature and pH were determined between 20 and 50 ꢀC and
pH 7 to 9. Activation energy was calculated using
activities measured below 35 ꢀC. The amounts of
coupling enzymes were not limiting under any assay
conditions used in this study. MTR-1-P isomerase was
also assayed by measuring the amount of MTRu-1-P

5-Methylthioribose 1-Phosphate Isomerase of Bacillus
2023
H. vulgare
(IDI2)
A
S. cerevisiae (Ypr118wp)
A. thaliana 1
H. sapiens 1
C. elegans 1
L. major
B. subtilis (MtnA)
T. maritima MSB8
A. fulgidus DSM 4304
T. kodakarensis KOD1 (E2B2)
C. elegans 2
eIF2Bα-LP
eIF2Bα
H. sapiens 2
A. thaliana 2
S. cerevisiae (GCN3)
0.1
B
B. subtilis (MtnA)
----------------------------------------------------------------------------------------------------
----------------------------------------------------------------------------------------------------
----------------------------------------------------------------------------------------------------
----------------------------------------------------------------------------------------------------
MRFFFFAVAGWGGGGNPVRCTLPPVRDAVVHFLSTFLCLSSLHFDVLRSFINRRPTLPKPSPPHNPTQPQGKRRTCETHRSAQRHLPIQKKKSLLKTHPA
----------------------------------------------------------------------------------------------------
S. cerevisiae (Ypr118wp)
T. maritima MSB8
A. fulgidus DSM4304
L. major
T. kodakarensis KOD1 (E2B2)
B. subtilis (MtnA)
1
---------------------------------MTHSFAVPRSVEWKETAITILNQQKLPDETEYLELTTKEDVFDAIVTLKVRGAPAIGITAAFGLALA
---------------------------------MSLEAIVFDRSEPENVSVKVLDQLLLPYTTKYVPIHTIDDGYSVIKSMQVRGAPAIAIVGSLSVLTE
------------------------------------MKLKTKTMEWSGNSLKLLDQRKLPFIEEYVECKTHEEVAHAIKEMIVRGAPAIGVAAAFGYVLG
----------------------------------------MRSIFWDDG-LKLIDQTKLPEKLEVIECRNVEELADAIKKLAVRGAPALEAAGAYGIALA
TSSEGFLARCLYLTTSTTTPLNPSLLALSTVMMSKPHHATLESIKYTPGSLRLLDQRKLPLETVFDDVLTVEDIWSAIKEMRVRGAPAIAVSAALGIAVA
----------------------------------------------------------------MAVVKEVLEIAEKIKNMEIRGAGKIARSAAYALQLQ
S. cerevisiae (Ypr118wp)
T. maritima MSB8
A. fulgidus DSM4304
L. major
T. kodakarensis KOD1 (E2B2)
B. subtilis (MtnA)
6 8 ------------AKDIETDNVTEFRRRLEDIKQYLNSSRPTAINLSWALERLSHSVENAI----SVNEAKTNLVHEAIQIQVEDEETCRLIGQNALQLFK
VQLIKHNPTSDVATLYSLVNWESTKTVLNKRLDFLLSSRPTAVNLSNSLVEIKNILKSSS----DLKAFDGSLYNYVCELIDEDLANNMKMGDNGAKYLI
------------LRDYKTG---SLTDWMKQVKETLARTRPTAVNLFWALNRMEKVFFENA----DRENLFEILENEALKMAYEDIEVNKAIGKNGAQLIK
------------AREREFADVDELKEHLKKAADFLASTRPTAVNLFVGIERALNAALKGE----SVEEVKELALREAEKLAEEDVERNRKMGEYGAELLE
TQRK--------AANGELKSGREVQTFLLTSCDFVMTSRPTAVNLFNCLRDLKAQVDKLDPT-KAAAEVAQAFVELAEAVYTNDVAFNEGIMRHGAAHIL
------------AEKSKATNVDEFWKEMKQAAKILFETRPTAVSLPNALRYVMHRGKIAYSSGADLEQLRFVIINAAKEFIHNSEKALERIGEFGAKRIE
S. cerevisiae (Ypr118wp)
T. maritima MSB8
A. fulgidus DSM4304
L. major
T. kodakarensis KOD1 (E2B2)
Glu193 Arg195
B. subtilis (MtnA)
1 5 2 KGD---------RIMTICNAGSIATSRYGTALAPFYLAKQK-----------------DLGLHIYACETRPVLQGSRLTAWELMQGGIDVTLITDSMAAH
DVLQKDGFKDEFAVLTICNTGSLATSGYGTALGVIRSLWKDSLAKTDKADSGLDNEKCPRMGHVFPLETRPYNQGSRLTAYELVYDKIPSTLITDSSIAY
DGS---------TILTHCNAGALATVDYGTALGVIRAAVES-----------------GKRIRVFADETRPYLQGARLTAWELMKDGIEVYVITDNMAGW
DGD---------VVLTYCNAGRLATVDWGTALGVVRSAVEQ-----------------GKEIRVIACETRPLNQGSRLTCWELMEDGIDVTLITDSMVGI
AAAKAE-GRDKVSILTICNTGALATSRYGTALGVVRQLFYD-----------------GKLERVYACETRPWNQGARLTVYECVQEDIPCTLICDGAASS
DGD---------VIMTHCHS--------KAAISVMKTAWEQ-----------------GKDIKVIVTETRPKWQG-KITAKELASYGIPVIYVVDSAARH
S. cerevisiae (Ypr118wp)
T. maritima MSB8
A. fulgidus DSM4304
L. major
T. kodakarensis KOD1 (E2B2)
Asp240
B. subtilis (MtnA)
2 2 6 TMKEK--QISAVIVGADRIAKNGDTANKIGTYGLAILANAFDIPFFVAAPLSTFDTKVKCGADIPIEERDPEEVRQ--ISGVRTAPSN------------
RIRTSPIPIKAAFVGADRIVLNGDTANKIGTYSLAVLAKRNNIPFYVAAPVSTIDPTIRSGEEIPIEERRPEEVTH--CGGTVINPENGSLILNESGEPI
LMKRG--LIDAVVVGADRIARD-AVFNKIGTYTVSVVAKHHNIPFYVAAPKATFD-WERTAKDVVIEERPREELIF--CGGNRIAPEG------------
VMQKG--MVDKVIVGADRIVQNGDTANKIGTYNLAVSAKFHGVKLYVAAPTTTLDVKTASGNHVEIEEREPTEITTNLVTKRQIAPLN------------
LMLNR--KIDAVVVGADRICRNGDTANKIGTLQLAVICKQFGIKFFVVAPKTTIDNVTETGDDIIVEERNPEEFKV--VTKQRVVADGP-----------
YMKMT----DKVVMGADSITVNGAVINKIGTALIALTAKEHRVWTMIAAETYKFHPETMLGQLVEIEMRDPTEVIP--EDELKTWPKN------------
S. cerevisiae (Ypr118wp)
T. maritima MSB8
A. fulgidus DSM4304
L. major
T. kodakarensis KOD1 (E2B2)
B. subtilis (MtnA)
3 1 0 ----------VPVFNPAFDITPHDLIS-GIITEKGIMTGNYEEEIEQLFKGEKVH-------
TGKVGIAPLEINVWNPAFDITPHELID-GIITEEGVFTKNSSGEFQLESLF-----------
----------VKVLNPAFDVTENTLIT-AIITEKGVIRPPFEENIKKILEV-----------
----------VKVYNPAFDPTPLENVT-ALITEYGVIYPPYEVNVPKVLKF-----------
---------HLSIWNPVFDITPSELITGGIITEKGVQAPAASAPYYDIASIIAQA-------
----------IEVWNPAFDVTPPEYVD-VIITERGIIPPYAAIDILREEFGWALKYTEPWED
S. cerevisiae (Ypr118wp)
T. maritima MSB8
A. fulgidus DSM4304
L. major
T. kodakarensis KOD1 (E2B2)
Fig. 2. Homology between MTR-1-P Isomerase and Related Proteins.
A, The deduced amino acid sequence of B. subtilis MTR-1-P isomerase (MtnA, NP 389238) was compared with sequences for several MtnA
homologs, Homo sapiens (H. sapiens1, NP 001026897), Arabidopsis thaliana (A. thaliana1, At2g05830), Hordeum vulgare subsp. vulgare
(BAB21393), Caenorhabditis elegans (C. elegans1, NP 506714), Saccharomyces cerevisiae (Ypr118wp, NP 015443), Leishmania major
(CAJ09465), Thermotoga maritima MSB8 (NP 228719), Archaeoglobus fulgidus DSM 4304 (NP 069206) and Thermococcus kodakarensis
KOD1 (YP 182598), and those of the alpha subunits of the eukaryotic initiation factor 2B in H. sapiens (H. sapiens2, NP 001405), A. thaliana
(A. thaliana2, At1g72340), C. elegans (C. elegans2, NP 499106), and S. cerevisiae (GCN3). The phylogenetic tree was produced by
CLUSTALW (http://www.ddbj.nig.ac.jp/search/clustalw-j.html) and TreeView (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) program.
B, Multiple alignments of sequences underlined in A. Identical and similar amino acid residues are shaded in black and gray, respectively. The
sequences are numbered according to that for the B. subtilis sequence. Dimer-forming residues are indicated by open triangles, putative proton-
subtracting residue by filled triangle. Alignment was displayed with BOXSHADE (http://www.ch.embnet.org/software/BOX form.html)
program.

2024
Y. SAITO et al.
Table 1. Molecular and Catalytic Properties of Recombinant MTR-
1-P Isomerase
product formed using the ferricyanide reducing sugar
assay16) and cysteine-carbazole17) methods.
Parameter
Value or description
Gel filtration for molecular mass determination. A
Superose 6 10/300 GL column (Amersham Pharmacia)
equilibrated with 50 mM Tris–HCl buffer (pH 8.0)
containing 1 mM MgCl2 and 150 mM NaCl was used
for molecular mass determination. The same buffer was
used for development of the column. The following
proteins from the Gel Filtration Calibration kit (Amer-
sham Pharmacia) were used for calibration of the
column: ferritin (440 kDa), aldolase (158 kDa), ovalbu-
min (43 kDa), and ribonuclease A (13.7 kDa).
Vmax
Km
20:4 ꢃ 0:8 mmol minꢂ1 mg proteinꢂ1b
b
138 ꢃ 9 mM
kcat
13 secꢂ1 siteꢂ1
8.1
37 ꢀC
68.7 kJ molꢂ1
Not needed
6.0
Optimum pH
Optimum temperature
Activation energy
Metal
Equilibrium constanta
Native molecular mass
Subunit molecular mass
Suggested composition
76 kDa
38.9 kDa
Homodimer
a[MTRu-1-P]/[MTR-1-P]
bMean ꢃ SE from three independent measurements
NMR analysis of the MtnA reaction product. The
NMR spectrum was recorded at 298 K in a Bruker DRX-
800 spectrometer operated at 800 MHz.7) MTR-1-P was
lyophilized for 16 h, dissolved in phosphate buffer made
with unopened 99.9% D2O solution (Wako) and used as
the starting substrate. H2O in the buffer used for
purification of recombinant enzyme was replaced with
D2O by passage through a NAP-5 column (Amersham
Pharmacia) equilibrated with the D2O-phosphate buffer.
decreased sharply above or below this pH and temper-
ature. The activation energy, Ea, of the isomerase
reaction was calculated from the Arrhenius plot to be
68.7 kJ molꢂ1 at pH 8.1. Isomerase activity increased in
a MTR-1-P concentration-dependent manner, and Vmax
and Km were 20.4 mmol minꢂ1 mg proteinꢂ1 and 138 mM
for MTR-1-P, respectively. The maximum activity
corresponded to 13 turnovers sꢂ1 siteꢂ1. The specificity
of the enzymatic reaction (kcat=Km) was about 9:3 ꢁ
Mass spectrometry of the reaction product of MTR-1-
P isomerase. Ribose 5-phosphate (R5P, Sigma-Aldrich,
St. Louis, MO) or lyophilized MTR-1-P was dissolved
in 50 mM Tris–HCl buffer (pD 8.0) prepared with 99.9%
D2O (Wako) and used as the substrate. R5P isomerase
(Sigma-Aldrich) was dissolved in the Tris–HCl buffer
(pD 8.0) containing 1 mM MgCl2. MTR-1-P isomerase
purified as above was passed through an NAP-5 column
(Amersham Pharmacia, USA) equilibrated with 50 mM
Tris–HCl buffer (pD 8.0). The isomerase reaction of
R5P and MTR-1-P isomerases, both at 10 mg in 100-ml
reaction mixture, proceeded in the presence of 1 mM
R5P and MTR-1-P respectively for 1 h at 37 ꢀC, and was
stopped by cooling to 4 ꢀC and removal of proteins with
Centricon YM-3 vial (Millipore, Billerica, MA) at 4 ꢀC.
The protein-free reaction mixture was lyophilized and
dissolved in Milli-Q water to analyze the molecular
mass of the reaction product with an ESI ion-trap mass
spectrometer (Bruker Daltonics, Bremen, Germany).
104 Mꢂ1 sꢂ1
.
The requirement of B. subtilis MTR-1-P isomerase
for a metal ion was analyzed by incubating the purified
enzyme with 100 mM EDTA in the presence of 50 mM
Tris–HCl buffer (pH 8.0) for 10 hr at 4 ꢀC and measur-
ing the remaining activity by the ferricyanide reducing
sugar assay and cysteine-carbazole methods.16,17) The
activity was not at all influenced by EDTA treatment,
indicating that the Bacillus MTR-1-P isomerase does not
require a metal ion for its catalysis.
To measure the equilibrium constant for the isomer-
ization reaction between MTR-1-P and MTRu-1-P, the
reaction of 1 mg of MTR-1-P isomerase protein and
15 mmol of MTR-1-P was monitored for 4 h by ion
chromatography equipped with a CarboPac PA1 col-
umn. The reaction was completed in 40 min and the
reaction products at equilibrium after 4 h were measured
by their phosphate contents (Fig. 3). MTR-1-P and
MTRu-1-P in the reaction mixture were present at
14:3 ꢃ 0:6 and 85:7 ꢃ 4:8% respectively of total phos-
phates applied to the column. The ratio was constant
over the reaction time for 4 h, indicating that the
equilibrium constant [MTRu-1-P]/[MTR-1-P] is 6.0
(Table 1). Indeed, 1H-NMR of the reaction mixture
revealed that most of the proton peaks were from
MTRu-1-P (Fig. 4), but not from the substrate MTR-1-
P, considered from the measured equilibrium constant of
the reaction. The peaks were well defined at 2.09 (m,
Results
There have been no previous reports of MTR-1-P
isomerase from any source been analyzed enzymolog-
ically. Here we determined the general physicochemical
and enzymological properties of the B. subtilis protein
before analyzing its reaction mechanism (Table 1). The
molecular mass of the purified active enzyme was found
to be 76 kDa by gel filtration. Since the molecular mass
of the subunit was deduced to be 38.9 kDa from its gene
sequence (Fig. 2B), the enzyme is probably composed
of two subunits.
2
3
3H, 6-CH3), 2.64 (dd, JHH ¼ 14:1 Hz, JHH ¼ 8:3 Hz,
2
3
5-CH2), 2.73 (dd, JHH ¼ 14:1 Hz, JHH ¼ 4:5 Hz, 5-
3
The activity of the forward reaction from MTR-1-P to
MTRu-1-P was maximal at pH 8.1 and 35 ꢀC, and
CH2), 4.11 (dt, JHH ¼ 8:3, 4.8 Hz, 4-CH), 4.47 (d,
3JHH ¼ 5:0 Hz, 3-CH), 4.64 (dd, JHH ¼ 18:9 Hz,
2

5-Methylthioribose 1-Phosphate Isomerase of Bacillus
2025
α
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
2-
OPO3
MTR-1-P
MTRu-1-P
ε
H
C
C
C
C
H
O
ε
δ
γ
H
H
OH
OH
β
α
CH2
SCH3
MTRu-1-P
δ
ε
β
γ
0
5
10
15
Retention time (min)
Fig. 3. Determination of the Equilibrium Constant of the MTR-1-P
Isomerase Reaction.
6.0
5.5
5.0
4.5
4.0
ppm
3.5
3.0
2.5
2.0
After incubation of MTR-1-P with MtnA for 4 h, the reaction
product was separated by CarboPac PA1 column. The phosphate
concentration was determined with a phosphate assay kit.
Fig. 4. 1H-NMR Spectrum of the Reaction Product Generated by
MTR-1-P Isomerase in the Presence of Buffer Prepared with 99.9%
D2O.
The reaction for the NMR spectrum was performed in D2O-
phosphate buffer to achieve equilibrium. The spectrum and the
assigned structure of the reaction product are displayed. The inset is
the partial, expanded spectrum around the peaks " from the C1
protons. The amount of protein added for NMR was 80 mg for MtnA.
2
3JHP ¼ 6:3 Hz, 1-CH2), and 4.70 ppm (dd, JHH ¼ 18:9
3
Hz, JHP ¼ 6:0 Hz, 1-CH2).
In the NMR spectrum, two of the C1 peaks were split
into double doublets caused by J couplings with hydro-
gen and phosphorus nuclei in the NMR spectrum
(Fig. 4, inset), indicating that C1 has two protons. This
suggests that the isomerase reaction of MTR-1-P
isomerase proceeds without incorporation of deuterium
from the medium into the product. Many sugar and
sugar phosphate isomerases show the enediol mecha-
nism, in which enediol is formed between two carbons
involved in the isomerization and a proton in the
produced sugar is from the medium.18–21) These facts
indicate the possibility that MTR-1-P isomerase pro-
ceeds in its reaction by another mechanism, not by the
enediol mechanism. To confirm this, the mass of the
reaction product formed from MTR-1-P by B. subtilis
MTR-1-P isomerase in D2O buffer was measured
(Table 2). The percentage of the intensity of (mono-
isotopic molecular mass + 1) to that of the monoiso-
topic molecular mass of MTRu-1-P formed by MTR-1-P
isomerase in the presence of 99.9% of D2O was 9%.
This value is very similar to the theoretical value (7.7%)
and to that of the substrate (7%) (Table 2). On the other
hand, the percentage of the reaction product of R5P
isomerase was 32%, while that of the substrate R5P was
6%. This discrepancy clearly demonstrates that a proton
is incorporated from the medium into the product in the
R5P isomerase reaction which proceeds in its reaction
by the enediol mechanism.18–21) It is known that some
protons in the product formed through an enediol are
from the medium even in isomerases that show the
enediol mechanism and that the frequency of the
incorporation of the proton from medium changes from
enzyme to enzyme. For example, the frequency at which
triose-phosphate isomerase transfers the proton on C2 to
C1 is only 2% of the total isomerization reaction, while
in the phosphoglucoisomerase reaction it is 50%.12)
Considering that an almost negligible level of incorpo-
ration of deuterium from the medium is observed (Fig. 4
and Table 2), B. subtilis MTR-1-P isomerase catalyzes
the isomerase reaction without adopting its enediol form
as a reaction intermediate.
Discussion
MtnA and its related proteins comprise a large family
Table 2. Percentage of Molecular Mass Plus One after Reaction in D2O Buffer
Sample
Relative intensity of MM +1 (%)a
MTR-1-P (no enzyme)
MTR-1-P + MtnA
7
9
Ribose-5-P (no enzyme)
Ribose-5-P + ribose-5-P isomerase
6
32
aThe intensity of the monoisotopic molecular mass (MM) normalized to 100.

2026
Y. SAITO et al.
CH3SH2C
H
H
CH3SH2C
H
O
O
H
H
H
H
O
hydride transfer
OPO3
2-
2-
OPO3
H
HO
OH
HO
O
HO
O
O
Asp240 ?
Asp240 ?
H
H
CH3SH2C
H
CH3SH2C
H
H
O
O
H
H
H
H
2-
2-
OPO3
O
OPO3
O
HO
HO
O
HO
O
O
Asp240 ?
Asp240 ?
Fig. 5. Proposed Reaction Mechanism of MTR-1-P Isomerase.
See the text for details.
in nature and are widely distributed among organisms
from bacteria to higher eukaryotes. The family is
divided into two clades in the phylogenetic tree
(Fig. 2A); one is for eIF2Bꢀ and the other for eIF2Bꢀ-
LP including MtnA. As well as MtnA, Ypr118wp of
yeast has been identified as an MTR-1-P isomerase.22)
Although eIF2Bꢀ-LPs of Leishmania major (PDB ID,
2A0U), Thermotoga maritima MSB8 (PDB ID, 1T9K),
Archaeoglobus fulgidus DSM4303 (Protein Data Bank;
PDB ID, 1T5O) have very similar protein structures to
that of yeast Ypr118wp (PDB ID, 1W2W), the functions
of these proteins and eukaryotic eIF2Bꢀ-LPs in Caeno-
rhabditis elegans, Arabidopsis thaliana, Hordeum vul-
gare, and Homo sapiens have not been analyzed.
However, IDI2, an eIF2Bꢀ-LP in H. vulgare, involved
in the synthesis of mugineic acid together with MtnD
Glu-193 and Arg-195 (the amino acid residues are
numbered according to the sequence in the B. subtilis
enzyme, Fig. 2B).
The equilibrium constant of the isomerase reaction
between MTR-1-P and MTRu-1-P was 6, favoring the
formation of the latter sugar phosphate (Table 1). The
free energy change of the reaction of MTR-1-P to
MTRu-1-P at 35 ꢀC was calculated to be 4.6 kJ molꢂ1
,
indicating that the free energy of MTR-1-P is slightly
smaller than that of MTRu-1-P.
Two mechanisms have been proposed for the aldose-
ketose isomerase reaction; the cis-enediol(ate) and
hydride transfer mechanisms.18) In the cis-enediol(ate)
mechanism, a proton is abstracted from a carbon of the
substrate by a base residue to make a cis-enediol(ate),
and a proton from another base residue of the enzyme
protein is donated to the counterpart carbon of the
enediol(ate). Since these protons are in equilibrium with
the protons of the water medium and are easily
exchanged with deuterium in D2O, the reaction product
must incorporate the deuterium atom from the medi-
um.19,20) The other mechanism is the hydride transfer
mechanism, in which a hydride on a carbon is directly
transferred to the neighboring cationic carbon.18,23,24) In
the isomerase reaction of B. subtilis MTR-1-P isomer-
ase, the product of the enzyme reaction in the presence
of 99.9% D2O showed no increase in mass after the
(1,2-dihydroxy-3-keto-5-methylthiopentene
dioxyge-
nase) in MSP, might be an MTR-1-P isomerase.8)
E2B2, an eIF2Bꢀ-LP in T. kodakaraensis, is RBP
isomerase.11) These considerations suggest that
eIF2Bꢀ-LPs might be involved in the isomerase reaction
of aldose-1-phosphate.
B. subtilis MtnA functioned as a dimmer enzyme
(Table 1). Ypr118wp of yeast and eIF2Bꢀ-LPs of
L. major, T. maritima and A. fulgidus have been iden-
tified as homodimers from structural analyses.22) All of
these homologues conserve the dimer-forming residues

5-Methylthioribose 1-Phosphate Isomerase of Bacillus
2027
microarray analysis of gene expression during Fe-
deficiency stress in barley suggests that polar transport
of vesicles is implicated in phytosiderophore secretion in
Fe-deficient barley roots. Plant J., 30, 83–94 (2002).
4) Christopher, S. A., Diegelman, P., Porter, C. W., and
Kruger, W. D., Methylthioadenosine phosphorylase, a
gene frequently codeleted with p16cdkN2a/ARF, acts as a
tumor suppressor in a breast cancer cell line. Cancer
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5) Riscoe, M. K., Ferro, A. J., and Fitchen, J. H., Analogs
of 5-methylthioribose, a novel class of antiprotozoal
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(1988).
reaction, in contrast to the case with R5P isomerase
(Table 2), and was virtually deuterium-free in NMR
analysis (Fig. 4, inset). These features of the reaction
were similar to those of xylose isomerase, which adopts
the hydride transfer mechanism.18,24) These results
suggest that B. subtilis MTR-1-P isomerase follows the
hydride transfer mechanism in its isomerization reac-
tion. An interesting point, unique to B. subtilis MTR-1-P
isomerase, is that this enzyme catalyzed the reaction
without the participation of a metal ion in the reaction
(Table 1).
A possible isomerase reaction is depicted in Fig. 5. To
start the isomerase reaction, the enzyme must abstract
the O2 proton using its base residue, and O2 and C2
form a carbonyl group by transferring O2 electrons to
C2. A hydrogen atom in the form of a hydride then shifts
from C2 to C1 with ꢁþ. The proton abstracted from O2
initially is backed to O5 to open the ring structure and
form MTRu-1-P. The possibly proton-abstracting resi-
due might be Asp240, considering the location of this
residue in the crystal of Ypr118wp of yeast.22) This
residue is conserved in T. kodakaraensis RBP isomerase
and other eIF2Bꢀ-LPs (Fig. 2B). In the MtnA structure
predicted by SWISS-MODEL software (http://
swissmodel.expasy.org//SWISS-MODEL.html) using
eIF2Bꢀ-LP of T. maritima (1T9K) as the template,
Asp240 is located in the same position as those in
Ypr118wp and eIF2Bꢀ-LPs from L. major, T. maritima,
and A. fulgidus (data not shown). We must wait for more
precise structural analysis of crystals of substrate-bind-
ing MTR-1-P isomerase to enable further discussion.25)
6) Furfine, E. S., and Abeles, R. H., Intermediates in the
conversion of 50-S-methylthioadenosine to methionine in
Klebsiella pneumoniae. J. Biol. Chem., 263, 9598–9606
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Ogasawara, N., and Yokota, A., A functional link
between RuBisCO-like protein of Bacillus and photo-
synthetic RuBisCO. Science, 302, 286–290 (2003).
8) Yamaguchi, H., Nakanishi, H., Nishizawa, N. K., and
Mori, S., Isolation and characterization of IDI2, a new
Fe-deficiency-induced cDNA from barley roots, which
encodes a protein related to the ꢀ subunit of eukaryotic
initiation factor 2B (eIF2Bꢀ). J. Exp. Bot., 51, 2001–
2007 (2000).
9) Webb, B. L., and Proud, C. G., Eukaryotic initiation
factor 2B (eIF2B). Int. J. Biochem. Cell Biol., 29, 1127–
1131 (1997).
10) Proud, C. G., eIF2 and the control of cell physiology.
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Acknowledgments
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quantitation of microgram quantities of protein utilizing
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13) Laemmli, U. K., Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature,
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14) Sekowska, A., Mulard, L., Krogh, S., Tse, J. K., and
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The authors thank Dr. Antoine Danchin, Pasteur
Institute, and Dr. Jun-ichi Azuma, Kyoto University, for
helpful discussion. We appreciate the help of Ms. Junko
Tsukamoto and Ms. Yoshiko Nishikawa in MS analysis.
This work was supported in part by a Grant-in-Aid
(No. 17208031) for Scientific Research from the Japan
Society for the Promotion of Science (JSPS), and in part
by a grant (FY2004-2006) for General Science and
Technology from the Asahi Glass Foundation, both to
A.Y.
´
´
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