Molecular characterization of a thermostable l-fucose isomerase from Dictyoglomus turgidum that isomerizes l-fucose and d-arabinose

Article abstract of DOI:10.1016/j.biochi.2012.05.009

A recombinant thermostable l-fucose isomerase from Dictyoglomus turgidum was purified with a specific activity of 93 U/mg by heat treatment and His-trap affinity chromatography. The native enzyme existed as a 410 kDa hexamer. The maximum activity for l-fucose isomerization was observed at pH 7.0 and 80 °C with a half-life of 5 h in the presence of 1 mM Mn²⁺ that was present one molecular per monomer. The isomerization activity of the enzyme with aldose substrates was highest for l-fucose (with a k_cat of 15,500 min^-1 and a K_m of 72 mM), followed by d-arabinose, d-altrose, and l-galactose. The 15 putative active-site residues within 5 A of the substrate l-fucose in the homology model were individually replaced with other amino acids. The analysis of metal-binding capacities of these alanine-substituted variants revealed that Glu349, Asp373, and His539 were metal-binding residues, and His539 was the most influential residue for metal binding. The activities of all variants at 349 and 373 positions except for a dramatically decreased k_cat of D373A were completely abolished, suggesting that Glu349 and Asp373 were catalytic residues. Alanine substitutions at Val131, Met197, Ile199, Gln314, Ser405, Tyr451, and Asn538 resulted in substantial increases in K_m, suggesting that these amino acids are substrate-binding residues. Alanine substitutions at Arg30, Trp102, Asn404, Phe452, and Trp510 resulted in decreases in k_cat, but had little effect on K_m.

Full text of DOI:10.1016/j.biochi.2012.05.009

Biochimie 94 (2012) 1926e1934
Contents lists available at SciVerse ScienceDirect
Biochimie
journal homepage: www.elsevier.com/locate/biochi
Research paper
Molecular characterization of a thermostable
L
-fucose isomerase
from Dictyoglomus turgidum that isomerizes
L
-fucose and -arabinose
D
*
Seung-Hye Hong, Yu-Ri Lim, Yeong-Su Kim, Deok-Kun Oh
Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A recombinant thermostable L-fucose isomerase from Dictyoglomus turgidum was purified with a specific
Received 5 January 2012
Accepted 11 May 2012
Available online 22 May 2012
activity of 93 U/mg by heat treatment and His-trap affinity chromatography. The native enzyme existed
as a 410 kDa hexamer. The maximum activity for
-fucose isomerization was observed at pH 7.0 and 80 ꢀ
with a half-life of 5 h in the presence of 1 mM Mn2þ that was present one molecular per monomer. The
isomerization activity of the enzyme with aldose substrates was highest for -fucose (with a kcat of
15,500 minꢁ1 and a Km of 72 mM), followed by
-arabinose, -altrose, and -galactose. The 15 putative
active-site residues within 5 Å of the substrate -fucose in the homology model were individually
replaced with other amino acids. The analysis of metal-binding capacities of these alanine-substituted
variants revealed that Glu349, Asp373, and His539 were metal-binding residues, and His539 was the
most influential residue for metal binding. The activities of all variants at 349 and 373 positions except
for a dramatically decreased kcat of D373A were completely abolished, suggesting that Glu349 and
Asp373 were catalytic residues. Alanine substitutions at Val131, Met197, Ile199, Gln314, Ser405, Tyr451,
and Asn538 resulted in substantial increases in Km, suggesting that these amino acids are substrate-
binding residues. Alanine substitutions at Arg30, Trp102, Asn404, Phe452, and Trp510 resulted in
L
C
L
Keywords:
D
D
L
L-Fucose isomerase
L
Dictyoglomus turgidum
Characterization
Active-site residues
decreases in kcat, but had little effect on Km
.
Crown Copyright Ó 2012 Published by Elsevier Masson SAS. All rights reserved.
1. Introduction
isomerization reactions, catalyzing not only the isomerization of
fucose to -fuculose, but also that of -arabinose to -ribulose. Thus,
-fucose isomerase and -arabinose isomerase are the same type of
enzymes.
L-
L
D
D
Aldose isomerases have been used for the conversion of various
types of monosaccharides due to their broad substrate specificity.
L
D
Among the aldose isomerases,
arabinose isomerase [4e6], and
been extensively studied in many microorganisms, including
various mesophiles, thermophiles, and hayperthermophiles.
L
-rhamnose isomerase [1e3],
L
-
The use of thermostable enzymes has many advantages,
including high reaction velocities, resistance to chemical denatur-
ation, reduced risk of contamination, high substrate solubility, and
prevention of generating undesirable byproducts [16]. Although
D
-xylose isomerase [7e9] have
However,
only two bacterial strains, Escherichia coli [10,11] and Caldicellulo-
siruptor saccharolyticus [12]. -Arabinose isomerase (E.C. 5.3.1.3)
has been investigated in only three strains Bacillus pallidus [13],
Klebsiella pneumoniae [14], and Klebsiella aerogenes (also known as
Aerobacter aerogenes) [4,15]. These two enzymes perform the same
L
-fucose isomerase (E.C. 5.3.1.25) has been studied in
the biochemical properties of several
erases have been characterized [12,14,17], only one thermostable
fucose isomerase from C. saccharolyticus has been reported [12].
Thus, the discovery of other thermostable -fucose isomerases is
required for obtaining more effective enzymes.
The structures of -fucose isomerase from E. coli [10] and D-
L-fucose/D-arabinose isom-
L-
D
L
L
arabinose isomerase from B. pallidus [13] have been determined,
and their active-site residues have been proposed. However, the
exact active-site residues have never been confirmed using kinetic
analyses for the wild-type and variant enzymes.
Abbreviations: LB, Luria-Bertani; IPTG, isopropyl-b-D-thiogalactopyranoside;
SDSsulfate, sodium dodecyl; PAGE, polyacrylamide gel electrophoresis; PIPES,
piperazine-N,N0-bis(2-ethanesulfonic acid); EDTA, ethylenediaminetetraacetic acid;
EPPS, N-(2-hydroxyethyl)piperazine-N0-(3-propanesulfonic acid); MD, molecular
dynamics; ICP-MS, inductively coupled plasma mass spectrometry; AAS, atomic
absorption spectroscopy.
In this study, a thermostable L-fucose isomerase from the
hyperthermophile Dictyoglomus turgidum was cloned, and its
biochemical properties, such as optimal metal ions, pH, and
temperature, thermostability, and substrate specificity, were
investigated. The metal-binding and substrate-binding residues
* Corresponding author. Tel.: þ82 2 450 4118; fax: þ82 2 444 6176.
E-mail address: deokkun@konkuk.ac.kr (D.-K. Oh).
0300-9084/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2012.05.009

S.-H. Hong et al. / Biochimie 94 (2012) 1926e1934
1927
were identified by conducting sequence alignment and kinetic
analyses of the wild-type and variant enzymes for the predicted
active-site residues of the homology model.
a linear imidazole gradient ranging from 10 to 250 mM at a flow
rate of 1 mL/min. The active fraction was collected and dialyzed at
4
ꢀC for 12 h against 50 mM piperazine-N,N0-bis(2-ethanesulfonic
acid) (PIPES) buffer (pH 7.0). The resulting solution was used as
a purified enzyme. The purification step using the column was
carried out by a fast protein liquid chromatography system (Bio-
Rad, Hercules, CA, USA) in a cold room at 4 ꢀC. The wild-type and
2. Materials and methods
2.1. Bacterial strains, plasmid, and culture conditions
variant L-fucose isomerases from D. turgidum were expressed in
Genomic DNA from D. turgidum DSMZ 6724 (DSMZ, Braunsch-
weig, Germany), E. coli ER2566 (New England Biolabs, Herfordshire,
UK), and pET28a(þ) plasmid (Novagen, Madison, WI, USA) were
E. coli as fusion proteins with hexa-histidine tags at the N-termini.
2.4. Determination of molecular mass
used as the source of L-fucose isomerase gene, host cells, and
expression vector, respectively. The recombinant E. coli cells con-
taining
-fucose isomerase/pET28a(þ) gene were cultivated in
a 2000-mL flask containing 500 mL of Luria-Bertani (LB) medium
and 20
g/mL kanamycin at 37 ꢀC with shaking at 200 rpm. When
the optical density of bacteria reached 0.6 at 600 nm, isopropyl-
thiogalactopyranoside was added at a final concentration of 0.1 mM
The subunit molecular mass of L-fucose isomerase was exam-
L
ined by SDS-PAGE (12% gels) under denaturing conditions, using
the proteins of a pre-stained ladder (MBI Fermentas, Glen Burnie,
MD, USA) as reference proteins. All protein bands were stained with
Coomassie Blue for visualization. The molecular mass of the native
enzyme from D. turgidum was determined by gel-filtration chro-
matography using a Sephacryl S-300 preparative-grade column HR
16/60 (GE Healthcare). The purified enzyme solution of 10 mg/mL
from the elution of the His-trap HP affinity chromatography column
as above-described was applied to the column and eluted with
50 mM TriseHCl buffer (pH 7.5) containing 150 mM NaCl at a flow
rate of 1 mL/min. The column was calibrated with apoferritin
m
b-D-
to induce L-fucose isomerase expression, after which the culture
was incubated with shaking at 150 rpm at 16 ꢀC for 12 h as the
conditions for obtaining the highest expression level.
2.2. Gene cloning and site-directed mutagenesis of
isomerase
L-fucose
(443 kDa), b-amylase (200 kDa), albumin (66 kDa), and carbonic
The gene encoding for a putative
amplified by PCR using the genomic DNA isolated from D. turgidum
as a template. The sequence of the oligonucleotide primers used for
L
-fucose isomerase was
anhydrase (29 kDa) as reference proteins (GE Healthcare), and the
molecular mass of the native enzyme was calculated by comparison
with the migration lengths of reference proteins.
gene cloning was based on the DNA sequence of D. turgidum L-
fucose isomerase (GenBank accession number YP_002352328.1).
2.5. Effects of metal ions, pH, and temperature on
isomerase activity
L-fucose
Forward (50-CATATGAGTAGAATTTTTCCTATGAGAATTGGG-30) and
reverse
primers
(50-CTCGAGAGTTATTTTGCTACTTTCTTATA-
CAAAGG-30) were designed to introduce the NdeI and XhoI
restriction sites (underline), respectively. The PCR-amplified DNA
fragments were purified using the QIAquick gel extraction kit
(Qiagen, Hilden, Germany), inserted into the pET28a(þ) vector
digested with the same restriction enzymes, and ligated into the
NdeI and XhoI sites of pET28a(þ). The resulting plasmid was
transformed into E. coli ER2566 and plated on LB agar containing
One unit (U) of
amount of enzyme required to produce 1
min at 80 ꢀC and pH 7.0. Unless otherwise stated, the reaction was
L
-fucose isomerase activity was defined as the
m
mol of -fuculose per
L
performed in 50 mM PIPES buffer (pH 7.0) containing 10 mM D-
arabinose and 1 U/mL of enzyme in the presence of 1 mM Mn2þ at
80 ꢀC for 15 min. The metal ion-treated enzyme was prepared by
adding 1 mM metal ions, such as Co2þ, Mn2þ, Mg2þ, Ca2þ, Zn2þ
,
20
m
g/mL kanamycin. A kanamycin-resistant colony was selected,
Cu2þ, Fe2þ, or Ni2þ, to the purified enzyme after treatment with
10 mM ethylenediaminetetraacetic acid (EDTA), followed by dial-
ysis at 4 ꢀC for 16 h against 50 mM PIPES buffer (pH 7.0). To
and plasmid DNA from the transformant was isolated using
a plasmid purification kit (Promega, Madison, WI, USA). DNA
sequencing was carried out using a DNA analyzer (ABI Prism
3730xl, PerkineElmer, Waltham, MA, USA). Gene expression was
evaluated by both sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and enzyme activity assay. Site-
directed mutagenesis was performed using the QuikChange site-
directed mutagenesis kit and protocol (Stratagene, Beverly, MA,
USA).
investigate the effect of metal ions on the activity of L-fucose
isomerase from D. turgidum, the reactions were performed in
50 mM PIPES buffer (pH 7.0) containing each metal ion. To examine
the effects of pH and temperature on enzyme activity, the pH values
were varied from 6.5 to 8.5 using 50 mM PIPES buffer (pH 6.5e7.5)
and 50 mM N-(2-hyroxyethyl)piperazine-N0-(3-propanesulfonic
acid) (EPPS) buffer (pH 7.5e8.5) at 80 ꢀC, and temperatures were
varied from 65 to 90 ꢀC at pH 7.0. The influence of temperature on
enzyme stability was monitored as a function of incubation time at
temperatures ranging from 65 to 85 ꢀC at pH 7.0. The experimental
data for thermal deactivation of the enzyme were fitted to a first-
order curve, and the half-life of the enzyme was calculated using
SigmaPlot 10.0 software (Systat Software, San Jose, CA, USA).
2.3. Purification of L-fucose isomerase
Recombinant cells were harvested from the culture broth by
centrifugation at 6000 ꢂ g for 20 min at 4 ꢀC, washed twice with
0.85% NaCl, and then resuspended in 50 mM phosphate buffer (pH
7.0) containing 300 mM NaCl. The resuspended cells were dis-
rupted using a sonicator. The crude enzyme from disrupted cell was
heated at 70 ꢀC for 10 min and then cooled at 4 ꢀC to remove
denaturated E. coli proteins. The unbroken cells and denaturated
proteins were removed by centrifugation at 13,000 ꢂ g for 20 min
at 4 ꢀC, and the supernatant obtained was used as a crude extract.
The enzyme solution of the supernatant was applied to a His-trap
HP affinity chromatography column (GE Healthcare, Piscataway,
NJ, USA) equilibrated with 50 mM phosphate buffer (pH 7.0). The
bound protein was subsequently eluted in the same buffer with
2.6. Determination of the specific activities and kinetic parameters
of the wild-type and variant enzymes
The specific activity (U/mg) was defined as the produced
amount of ketose as a product per enzyme amount per unit reac-
tion time. Various concentrations of aldoses (10e250 mM) were
used to determine the kinetic parameters of the enzyme. The
reactions were performed in 50 mM PIPES buffer (pH 7.0) at 80 ꢀC
for 15 min. The kinetic parameters, Km (mM) and kcat (minꢁ1), were

1928
S.-H. Hong et al. / Biochimie 94 (2012) 1926e1934
determined by LineweavereBurk plot analysis derived from the
MichaeliseMenten equation. To calculate the kcat, the amount of
protein was divided by the total molecular mass.
(Fig. 1a), which was consistent with the calculated value of
68,363 Da based on the 606-amino acid residues plus a hexa-
histidine tag at the carboxyl terminus using the Compute pI/Mw
tool. The native enzyme existed as a hexamer with a molecular
mass of 410 kDa, as determined by gel filtration chromatography
(Fig. 1b).
2.7. Homology modeling and substrate docking
The sequence of L-fucose isomerase from D. turgidum was
submitted to the HHpred meta server (http://meta.bioinfo.pl/
submit_wizard.pl) for automated full-chain protein structure
prediction to construct the homology model. The 10 most probable
3.2. Effects of metal ions, pH, and temperature on the activity of
fucose isomerase from D. turgidum
L-
conformations were predicted for the
using the known E. coli structure, which had been determined in
a complex with the substrate -fucose (PDB code 1FUI), as
L-fucose isomerase monomer
L
-Fucose isomerases are metalloenzymes that require a divalent
ion metal cofactor, such as a Mn2þ, for sugar isomerization [10,12].
The metal ions in the purified enzyme obtained after treating His-
trap HP affinity chromatography were found to be the small
amounts of Mg2þ and Ca2þ by the analysis of ICP-MS. Therefore, the
metal ions in the purified enzyme should be removed to evaluate
correctly the effects of metal ions on the activity of the enzyme.
After the removal of metal ions by treatment with EDTA, the
activity of the putative
(Fig. 2a). The purified enzyme without EDTA treatment showed
M Mg2þ and 70
activity because the enzyme contained 3.5 mM
L
a template. The most compatible conformation having a mono-
meric arrangement and the suggested position of the metal ions
(Mn2þ) was selected. All of the structural modifications were per-
formed using the AMBER 11 program [18]. Several cycles of energy
minimization (CHARMM27 force field), a 14-Å cutoff for electro-
statics and van der Waal’s interactions, relaxation with temperature
(310 K), and symmetry-driven annealing were performed to opti-
mize the conformation. The modeled position of the backbone was
restrained during relaxation with a spring constant of 1 kcal/mol/Å2
applied to the backbone atoms to limit the possible deformation of
the backbone during the relaxation of steric conflicts between the
side chains of the homology model. The resulting displacement of
the backbone during relaxation was on the order of 0.1 Å. Atomic
L-fucose isomerase was almost abolished
m
charges and optimized conformation for linear L-fucose were
calculated with Gaussian 03 using the HF/6-31G(d) basis set [19].
Homology modeling was performed at the program facility of Korea
Research Institute of Bioscience and Biotechnology (KRIBB). Auto-
mated docking was carried out by the AutoDock 4.2 program [20].
The substrate orientation giving the lowest interaction energy was
chosen for additional rounds of docking.
2.8. Analytical methods
Protein concentrations were determined by the Bradford
method using bovine serum albumin as a standard protein [21]. The
metal ions of the purified enzyme were identified by an inductively
coupled plasma mass spectrometry (ICP-MS) (Elan 6100, Perkin
Elmer, Norwalk, CT, USA). The concentrations of Mn2þ for the EDTA-
treated enzyme were analyzed by an atomic absorption spectros-
copy (AAS) (Thermo Scientific iCE 3000 series, Perkin Elmer). The
concentrations of monosaccharides (Carbosynth, Newbury, Berk-
shire, UK; SigmaeAldrich, St. Louis, MO, USA) were determined by
a Bio-LC system (Dionex ICS-3000, Sunnyvale, CA, USA) equipped
with an electrochemical detector and a CarboPac PA1 column. The
column was eluted at 30 ꢀC with 200 mM sodium hydroxide at
a flow rate of 1 mL/min.
3. Results
3.1. Gene cloning, purification, and molecular mass of L-fucose
isomerase from D. turgidum
A gene of 1818 bp encoding for a putative L-fucose isomerase
from D. turgidum, with the same sequence as that reported in
GenBank (accession number YP_002352328.1), was cloned and
then expressed in E. coli. The expressed enzyme was active for
L-
Fig. 1. SDS-PAGE analysis and molecular mass determination of the L-fucose isomerase
from D. turgidum. (A) SDS-PAGE analysis of D. turgidum L-fucose isomerase from each
purification step. Lane 1, marker proteins; lane 2, crude extract; lane 3, supernatant
after heat treatment at 70 ꢀC for 10 min; lane 4, His-trap column product (purified
enzyme). (B) Determination of the total native molecular mass of D. turgidum L-fucose
fucose. The putative recombinant -fucose isomerase was purified
L
as a soluble protein from crude extract from E. coli by heat treat-
ment and His-trap affinity chromatography, with a final purifica-
tion of 50-fold, a yield of 62%, and a specific activity of 93 U/mg for
isomerase by gel filtration chromatography. Apoferritin (443 kDa),
b-amylase
L-fucose. The purified enzyme analyzed by SDS-PAGE showed
(200 kDa), albumin (66 kDa), and carbonic anhydrase (29 kDa) (C); D. turgidum L-
fucose isomerase (B).
a single band with a molecular mass of approximately 68 kDa

S.-H. Hong et al. / Biochimie 94 (2012) 1926e1934
1929
a
120
100
80
60
40
20
0
6.0
6.5
7.0
7.5
8.0
8.5
9.0
pH
b
120
100
80
60
40
20
0
60
65
70
75
80
85
90
95
o
Temperature ( C)
Fig. 3. Effects of pH and temperature on the activity of L-fucose isomerase from D. turgidum
for L-fucose. (A) pH effect. The buffers used in the pH experiments were 50 mM PIPES buffer
(pH 6.5e7.5) and EPPS buffer (pH 7.5e8.5). (B) Temperature effect. The reactions were
performed in 50 mM PIPES buffer (pH 7.0) containing 10 mM L-fucose. Data represent the
means of three experiments and error bars represent the standard deviations.
Fig. 2. Effect of metal ions on the activity of L-fucose isomerase from D. turgidum. (A)
Effect of metal ions on the activity of L-fucose isomerase from D. turgidum for L-fucose.
The reactions were performed in 50 mM PIPES buffer (pH 7.0) containing 10 mM L-
fucose and 1 mM of each metal ion at 80 ꢀC for 15 min. None indicates the purified
enzyme without EDTA treatment. (B) Effect of the Mn2þ concentration on L-fucose
isomerase activity. Data represent the means of three experiments and error bars
represent the standard deviations.
maximum activity was observed at 80 ꢀC. At temperatures of 75
and 85 ꢀC, the activity was approximately 80% of the maximum
(Fig. 3b). The thermal stability of the enzyme was examined by
measuring the enzyme activity over time at temperatures ranging
from 65 to 85 ꢀC. The enzyme displayed first-order kinetics for
thermal inactivation, with half-lives of 20, 12, 7, 5, and 2 h at 65, 70,
75, 80, and 85 ꢀC, respectively (Fig. 4).
Ca2þ in the reaction solution. The addition of Mn2þ, Mg2þ, Ca2þ
Zn2þ, Ni2þ, or Co2þ enhanced the activity of
-fucose isomerization,
whereas the addition of Fe2þ and Cu2þ inhibited the activity.
Among the metal ions tested, Mn2þ was the most effective for
,
L
L-
fucose isomerization in an aprroximately10-fold increase in activity
relative to the enzyme without EDTA treatment and Mn2þ addition.
The optimal Mn2þ concentration was 1 mM (Fig. 2b). AAS results
3.3. Substrate specificity of L-fucose isomerase from D. turgidum
demonstrated that EDTA-treated enzyme of the putative L-fucose
isomerase from D. turgidum did not contain Mn2þ, and the amount
of Mn2þ in Mn2þ-treated enzyme after EDTA treatment was
approximately 1 mol per mol of enzyme monomer. Although Ca2þ
and Mg2þ were detected in the purified enzyme, Mg2þ and Ca2þ
showed no crucial effect on the enzyme activity. However, Mn2þ
stimulated significantly the enzyme activity. Thus, Mn2þ-activated
enzyme was used in all the subsequent experiments.
The enzyme activity was examined over a pH range of 6.5e8.5 at
80 ꢀC. The maximum activity was observed at pH 7.0. At pH 6.5 and
7.5, the activity was approximately 80% of the maximum (Fig. 3a).
The effect of temperature on enzyme activity was assessed, and the
The specific activity of the putative
D. turgidum for monosaccharides was investigated with
forms of all pento-aldoses and hexo-aldoses. Among the substrate
aldoses, the specific activity was highest for -fucose, followed by
arabinose, -altrose, and -galactose. No enzyme activity was found
for the other pento-aldoses or hexo-aldoses. The
MichaeliseMenten constant (Km), turnover rate (kcat), and catalytic
efficiency (kcat/Km) of the enzyme for -fucose, -arabinose, and
altrose are shown in Table 1. Although the enzyme had a higher
affinity for -altrose than for -fucose and -arabinose, the isom-
erization rate for -altrose by the enzyme was slower than those for
L
-fucose isomerase from
D- and
L-
L
D-
D
L
L
D
D-
D
L
D
D

1930
S.-H. Hong et al. / Biochimie 94 (2012) 1926e1934
100
80
60
40
20
0
2
4
6
8
10
12
Time (h)
Fig. 4. Thermal inactivation of L-fucose isomerase from D. turgidum for L-fucose. The
enzymes were incubated at 65 (C), 70 (,), 75 (:), 80 (B), and 85 ꢀC (-). A portion
of the reaction mixture was withdrawn at each time interval and the relative activity
was determined. Data represent the means of three experiments and error bars
represent the standard deviations.
Fig. 5. Proposed structure of the active site in L-fucose isomerase from D. turgidum
complexed with the substrate L-fucose. The molecular model shows the active-site
residues, including metal-binding residues (white color), L-fucose (yellow color), and
Mn2þ (orange color). The white and red sticks represent L-fucose as a substrate. The
dashed line presents hydrogen bond. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
L-fucose and D-arabinose. The kcat/Km for L-fucose was higher than
for any other monosaccharide, indicating that this enzyme is an
fucose isomerase.
L-
N538A were 7, 13, 91, 8, and 21% of the wild-type enzyme activity,
respectively (Table 2). But, E349A showed no activity. Glu349 and
Asp373 were replaced with other respective typical amino acids,
including polar uncharged (Gln), negatively charged (Asp, Glu),
positively charged (Lys), and aromatic amino acids (Trp). All of the
3.4. Selection of active-site residues by molecular modeling
The homology model for the active-site of D. turgidum
isomerase was generated based on the known structure of the E.
coli -fucose isomerase (PDB code 1FUI) as a template. A ligand-
docking study with the linear form of the substrate -fucose was
L-fucose
L
L
conducted using this homology model to identify specific residues
involved in enzyme activity. The active-site residues within
a sphere of 5-Å radius centered on the docked substrate were
identified as Arg30, Trp102, Val131, Met197, Ile199, Gln314, Glu349,
Asp373, Asn404, Ser405, Tyr451, Phe452, Trp510, Asn538, and
His539 (Fig. 5). These 15 residues were selected as determinant
candidates for enzyme activity.
Table 2
Specific activities and kinetic parameters of the wild-type and variant
isomerases from D. turgidum for -fucose as a substrate.
L-fucose
L
Enzyme Specific activity
Km (mM)
kcat (minꢁ1
)
kcat/Km
(
m
mol minꢁ1 mgꢁ1
)
(mMꢁ1 minꢁ1
)
Wild-type 33 ꢃ 0.2
72 ꢃ 3.0
85 ꢃ 2.7
77 ꢃ 0.6
148 ꢃ 7
166 ꢃ 1
202 ꢃ 2
225 ꢃ 2
ND
ND
ND
ND
ND
15,500 ꢃ 20
3230 ꢃ 20
1150 ꢃ 14
215 ꢃ 9
R30A
3.4 ꢃ 0.1
2.5 ꢃ 0.2
27 ꢃ 0.2
2.4 ꢃ 0.16
4.3 ꢃ 0.06
0.93 ꢃ 0.03
ND
ND
ND
ND
ND
37 ꢃ 0.03
15 ꢃ 0.01
W102A
V131A
M197A
I199A
13,000 ꢃ 473 89 ꢃ 0.06
3.5. Specific activities and kinetic parameters of the variants of the
active-site residues for L-fucose isomerization
1070 ꢃ 1
2000 ꢃ 1
443 ꢃ 20
ND
ND
ND
6.5 ꢃ 0.04
9.8 ꢃ 0.1
2.0 ꢃ 0.1
ND
Q314A
E349A
E349D
E349Q
E349K
E349W
D373A
D373E
D373Q
D373K
D373W
N404A
S405A
Y451A
F452A
W510A
N538A
H539A
The selected 15 residues were individually replaced with
alanine, the wild-type and 15 alanine-substituted variant enzymes
were expressed as single bands on SDS-PAGE (data not shown). The
specific activities of R30A, W102A, Q314A, E349A, D373A, N404A,
Y451A, W510A, and H539A for L-fucose isomerization were not
more than 4% of the wild-type enzyme activity, whereas the
ND
ND
ND
ND
ND
ND
0.47 ꢃ 0.01
140 ꢃ 5.8 480 ꢃ 16
3.4 ꢃ 0.05
ND
ND
ND
ND
ND
ND
ND
ND
ND
619 ꢃ 1
14,100 ꢃ 20
2460 ꢃ 10
1220 ꢃ 60
155 ꢃ 1
6890 ꢃ 25
202 ꢃ 6
ND
ND
ND
ND
ND
ND
ND
1.3 ꢃ 0.03
30 ꢃ 0.5
0.67 ꢃ 0.02
2.6 ꢃ 0.04
0.35 ꢃ 0.01
6.8 ꢃ 0.4
2.1 ꢃ 0.02
specific activities of V131A, M197A, I199A, S405A, Fe452A, and
92 ꢃ 0.2
385 ꢃ 3
302 ꢃ 10
86 ꢃ 4.0
90 ꢃ 0.7
339 ꢃ 3
52 ꢃ 0.6
6.7 ꢃ 0.02
37 ꢃ 0.3
8.1 ꢃ 0.3
14 ꢃ 0.9
1.7 ꢃ 0.02
20 ꢃ 0.2
3.9 ꢃ 0.03
Table 1
Specific activities and kinetic parameters of L-fucose isomerase from D. turgidum.
Substrate
Product
Specific
activity
(U mgꢁ1
Km (mM) kcat (minꢁ1
)
kcat/Km
(mMꢁ1 minꢁ1
)
)
L
-Fucose
-Arabinose
-Altrose
L
-Fuculose 93 ꢃ 1.0 90 ꢃ 0.2 38,700 ꢃ 10 430 ꢃ 1
ND not detected.
D
D
D
D
-Ribulose 33 ꢃ 0.2 72 ꢃ 3.0 15,500 ꢃ 20 215 ꢃ 9
-Psicose
23 ꢃ 0.1 67 ꢃ 0.1 10,200 ꢃ 10 152 ꢃ 0.3
ND ND
The reactions were performed in 50 mM PIPES buffer (pH 7.0) containing 10 mM L-
fucose at 80 ꢀC for 15 min. The reactions of R30A, W102A, D373A, and H539A were
L-Galactose
L-Tagatose 0.8 ꢃ 0.1 ND
performed with 5, 3, 10, and 10 mM Mn2þ, respectively, whereas the reactions of the
ND not detected.
other variants were performed with 1 mM Mn2þ
.
Data represent the means and standard deviations from 3 separate experiments.
Data represent the means and standard deviations from three separate experiments.

S.-H. Hong et al. / Biochimie 94 (2012) 1926e1934
1931
The optimum concentrations of Mn2þ for the variants were
determined because the affinity of the enzyme to Mn2þ might be
not the same. The optimum concentrations of Mn2þ for R30A,
W102A, D373A, and H539A were 5, 3, 10, and 10 mM, respectively
(Fig. 6), whereas those for the other variants were 1 mM (data not
Table 3
Amount of manganese in the wild-type and variant enzymes as determined by AAS.
Enzyme
Manganese,
ppm ( mol)
Total protein,
g ( mol)
Manganese
bound, % of
wild-type, (
m
m
m
m
mol
Mn2þ
/mmol enzyme)
shown). The kinetic parameters of the variants for L-fucose isom-
Wild-type
E349A
D373A
4.53 (83)
0.78 (15)
2.62 (48)
0.06 (1)
40 (85)
39 (83)
40 (85)
38 (81)
100 (0.98)
18 (0.18)
57 (0.56)
1 (0.01)
erization were investigated at their optimum concentrations of
Mn2þ (Table 2). The kcat values of V131A, S405A, and N538A were
approximately 84, 90, and 45% of the wild-type enzyme kcat
,
H539A
respectively, whereas those of the other alanine-substituted vari-
ants were less than 16%. The Km values of R30A, W102A, N404A,
F452A, W510A, and H539A were similar to that of the wild-type
enzyme, while the Km values of V131A, I199A, Q314A, D373A,
E349A, and N538A were 1.9- to 4.7-fold higher.
The term of ‘% of wild-type’ means the relative value for manganese binding of the
variant enzymes to that of the wild-type enzyme.
120
100
80
60
40
20
0
4. Discussion
The amino acid sequence of
L-fucose isomerase from D. turgidum
has 87, 71, 61, and 60% identities with
L
-fucose/ -arabinose isom-
D
erases from the hyperthermophile C. saccharolyticus [12] and the
mesophiles B. pallidus [13], E. coli [17], and K. pneumoniae [14],
respectively. These results indicate that the L-fucose isomerase
from the hyperthermophile D. turgidum is much more closely
related to that of the hyperthermophile C. saccharolyticus than to
those of the mesophiles.
The biochemical properties and kinetic parameters of
-arabinose isomerases are summarized in Table 4. The subunit
molecular masses of -fucose/ -arabinose isomerases were
65e68 kDa. -Fucose/ -arabinose isomerases existed as hexamers
L-fucose/
D
L
D
L
D
0
5
10
15
20
(Fig. 1b) [10,13] except for the enzyme from C. saccharolyticus,
which was a trimer [12].
2+
Mn concentration (mM)
Fig. 6. Effect of Mn2þ concentration on the activities of R30A (C), W102A (,), D373A
(-), and H539A (B) variant L-fucose isomerases from D. turgidum. variants.
The bound metal ion in
E. coli or B. pallidus obtained after adding Mn2þ was Mn2þ [10,13].
The metal ions found in -arabinose isomerase from B. pallidus
under physiological conditions were Mg2þ, Ca2þ, Naþ, and Kþ [13].
The metal ions in the purified -fucose isomerase from D. turgidum
were Mg2þ and Ca2þ. Although the amount of Mn2þ in Mn2þ
L-fucose/D-arabinose isomerase from
D
variant enzymes were expressed also as single bands on SDS-PAGE
(data not shown). All substitutions at these residues except for
alanine substitution at position 373 abolished activity. The amounts
of associated Mn2þ in E349A, D373A, and H539A were 17, 58, and
1% of that of the wild-type enzyme (Table 3).
L
-
treated enzyme was 1 mol/mol enzyme monomer, the total metal
content of Mg2þ and Ca2þ in the purified enzyme without EDTA
Table 4
Biochemical properties and kinetic parameters of
L-fucose/D-arabinose isomerases.
Enzyme Substrate Microorganism
Mr
Temperature pH
Specific activity Metal Km (mM)
(U mgꢁ1
Vmax (U mLꢁ1
)
Reference
(kDa) (ꢀC)
)
L
-Fucose isomerase
L
L
L
-Fucose
-Fucose
-Fucose
Escherichia coli B/r
Escherichia coli K-2
Caldicellulosiruptor
saccharolyticus
65
65
68
37
37
75
8.0 64 (69%)
8.0 63 (68%)
7.0 76 (82%)
Co2þ
Co2þ
Mn2þ
42 (47%)
45 (50%)
141 (157%) 0.50 (22%)
0.25 (11%)
0.26 (11%)
[17]
[17]
[12]
L-Fucose
Dictyoglomus turgidum 68
80
40
55
7.0 93 (100%)
9.3 38 (41%)
9.0 NR
Mn2þ
Mn2þ
Mn2þ
90 (100%)
51 (57%)
NR
2.3 (100%)
NR
NR
This study
[14]
[13]
D
-Arabinose isomerase
D
D
-Arabinose Klebsiella pneumoniae
-Arabinose Bacillus pallidus
65
66
NR not reported.
Table 5
Properties of D. turgidum
L
-fucose isomerase and other microbial
L-fucose/D-arabinose isomerases determined from a comparison of the deduced amino acid sequences.
Enzyme
Enzyme source
Theoretical pI
GRAVY index
Aliphatic index
Instability index
Percentage of
charged amino acids
(positive/negative)
D
-Arabinose isomerase
Klebsiella pneumoniae
Bacillus pallidus
Escherichia coli Kꢁ12
Caldicellulosiruptor
saccharolyticus
5.71
5.23
5.43
5.71
ꢁ0.228
ꢁ0.349
ꢁ0.260
ꢁ0.284
77.02
76.55
77.82
80.84
35.04 (stable)
29.18 (stable)
40.67 (unstable)
34.35 (stable)
9.5/11.7
11.4/13.8
9.7/12.6
L
-Fucose isomerase
11.7/13.2
Dictyoglomus turgidum
5.69
ꢁ0.253
82.82
33.97 (stable)
11.8/13.5
GRAVY, grand average of hydropathicity.

1932
S.-H. Hong et al. / Biochimie 94 (2012) 1926e1934
Fig. 7. Alignment of amino acid sequences of L-fucose isomerases. The GenBank accession numbers of L-fucose isomerases are as follows: CsFI (Caldicellulosiruptor saccharolyticus),
A4XJ56; DtFI (Dictyoglomus turgidum), B8E1T1; BpFI (Bacillus pallidus), AB429010; EcFI (Escherichia coli), EG10349; and KpFI (Klebsiella pneumoniae), A6TD83. Asterisk, colon, and
dot present identity, conserved substitutions, and semi-conserved substitution, respectively.

S.-H. Hong et al. / Biochimie 94 (2012) 1926e1934
1933
treatment was 0.02 mol/mol enzyme. These results indicate that
some metal ions such as Mg2þ and Ca2þ may be occupied randomly
in the enzyme and that excess Mn2þ is bound to the enzyme with
E. coli. Moreover, the proposed active-site residues of the homology
model of D. turgidum -fucose isomerase were completely
conserved with those of E. coli -fucose isomerase (Figs. 5 and 7).
Thus, the homology model based on the structure of E. coli -fucose
L
L
one binding site per a molecule. The most effective metal ion for
fucose isomerization of -fucose/
-arabinose isomerases was Mn2þ
except that -fucose isomerase from E. coli showed the highest
L-
L
L
D
isomerase (PDB code 1FUI) can be used for the identification of
active-site residues. Glu349, Asp373, and His539 in the homology
model were suggested to be metal-binding residues (Fig. 5). The
low amounts of associated Mn2þ in E349A, D373A, and H539A
indicated that Glu349, Asp373, and His539 were metal-binding
residues. The lowest amounts of associated Mn2þ in H539A,
which was 1% of that of the wild-type enzyme, meant that His539
was the most influential residue for metal binding (Table 3). The Km
of D373A at the optimal concentration of Mn2þ increased, sug-
gesting that Asp373 was involved in substrate binding. Glu349 and
Asp373 were previously suggested to be catalytic residues that
were directly involved in the aldose-ketose isomerization reaction
L
activity by adding Co2þ [17].
L-Fucose/D-arabinose isomerases from
E. coli, B. pallidus, and C. saccharolyticus were used as Mn2þ-acti-
vated enzymes for their characterization [10e13], and the bound
metal ion in the crystal structures of fucose/
D-arabinose isomerases
from E. coli and B. pallidus was Mn2þ [10,13]. Thus, Mn2þ-activated
enzyme was used in the characterization of D. turgidum L-fucose
isomerase.
The temperature for the maximum activity of
L-fucose isom-
erase from D. turgidum (Fig. 3) was highest among the character-
ized L-fucose/D-arabinose isomerases. The half-life of the enzyme at
80 ꢀC (Fig. 4) was 5.0-fold higher than that from C. saccharolyticus
[12]. These results indicated that this enzyme from D. turgidum is
by transferring a proton between O1 and O2 of L-fucose [13]. The
kcat of D373A was dramatically decreased and the activities of all
variants at 349 and 373 positions except for D373A were
completely abolished. These results suggest that Glu349 and
Asp373 are the critical catalysis residues for the isomerization.
Alanine substitutions at Val131, Met197, Ile199, Gln314, Ser405,
Tyr451, and Asn538 resulted in substantial increases in Km, sug-
gesting that these amino acids are substrate-binding residues
(Table 2). Val131, Met197, and Ile199 in the homology model are
the most thermostable enzyme among
erases. -Fucose isomerase from D. turgidum has the highest specific
activity, Vmax, and kcat/Km among the characterized -fucose/
arabinose isomerases, indicating that this newly characterized
enzyme is the most efficient for -fucose isomerization.
To evaluate the thermostability of -fucose isomerase from D.
L-fucose/D-arabinose isom-
L
L
D-
L
L
turgidum, physiochemical properties of the protein, including theo-
retical isoelectric point (pI), total percentage of positive and negative
residues, instability index, and aliphatic index [22], were compared
located opposite of the hydroxyl groups in the substrate
(Fig. 5). The hydroxyl group of Ser405 interacts directly with the
oxygen atom of -fucose, Gln314 and Asn538 make hydrogen bonds
with -fucose, and Tyr451 not only provides hydrophobic envi-
L-fucose
with those of other thermophilic and mesophilic
L-fucose/D-arabinose
L
isomerases (Table 5). The GRAVY index which stands for the hydro-
phobicity of protein did not provide outstanding rule between
thermophilic and mesophilic enzymes. However, the pI of D. turgidum
L
ronment but also functions in detaining the substrate in the
enzyme [10]. Alanine substitutions at Arg30, Trp102, Asn404,
Phe452, Trp510, and His539 resulted in decreases of kcat, but had
little effect on Km, which were resulted from structural alteration in
the active site. The Arg30, Trp102, and Val131 residues, which were
located at the interface of subunits, may serve as architectural
stabilizers and important bond managers. However, the kinetic
data suggest that Val131 are involved in substrate binding but
Arg30 and Trp102 are not involved in substrate binding.
L
-fucose isomerase was higher than those of mesophilic
isomerases except for K. pneumoniae -fucose isomerase because of
a higher content of basic amino acids and a lower content of acidic
amino acids. The aliphatic index of -fucose isomerase from D. tur-
gidum was highest among the characterized -fucose/ -arabinose
L-fucose
L
L
L
D
isomerases.Thethermostablefeatureoftheenzymemaybeshownby
the aliphatic index because the index wasregarded asa positive factor
for the increase of thermostability of globular proteins. The instability
In this study, a gene encoding the putative L-fucose isomerase
index of
L
-fucose isomerase from D. turgidum was calculated to be
from the hyperthermophile D. turgidum was cloned, expressed,
purified, and characterized. The enzyme had the most thermosta-
33.97. This value indicated that the enzyme was stable because
a protein with an instability index of less than 40 was assumed as
stable [23]. The content of charged amino acids in thermophilic
bility and the highest activity for
L-fucose among the characterized
L
-fucose/ -arabinose isomerases. The active-site residues of the
D
L
-fucose isomerases was higher than that in mesophilic enzymes
enzyme were identified by conducting kinetic analyses of the wild-
type and variant enzymes based on the established homology
model. Glu349, Asp373, and His539 were identified as metal-
binding residues. His539 was the most influential residue for
metal binding, and Glu349 and Asp373 were critical catalytic
residues. Val131, Met197, Ile199, Gln314, Ser405, Tyr451, and
Asn538 were characterized as substrate-binding residues, whereas
Arg30, Trp102, Asn404, Phe452, and Trp510 were activity-related
residues.
except for B. pallidus enzyme, and might generate a network of
intersubunit ion pairs or salt bridges to quaternary structure of the
enzymes, resulting in the increase of thermostability.
L-Fucose isomerases from D. turgidum (Table 1) and
C. saccharolyticus [12] have substrate specificity for the aldose
substrates that possess hydroxyl groups oriented in the left-handed
configuration at the C2 position and in the right-handed configu-
ration at the C3 and C4 positions, such as
altrose, and -galactose. The specific activity of D. turgidum
isomerase for -fucose and -altrose was 1.2- and 1.5-fold higher,
respectively than that of C. saccharolyticus -fucose isomerase,
whereas the specific activity of D. turgidum -fucose isomerase for
-arabinose and -galactose was 1.9- and 2.5-fold lower, respec-
tively. In contrast, -arabinose isomerase from K. pneumoniae
exhibited broad substrate specificity for -fucose, -arabinose,
altrose, -galactose, -xylose, -mannose, and -glucose [14].
L-fucose,
D
-arabinose,
D-
L
L-fucose
L
D
Acknowledgments
L
L
This work was supported by the National Research Foundation
of Korea (NRF) grant (No. 2010-0019306) and by the National
Research Lab. Program grant (R0A-2007-000-20015-0) of the
Ministry of Education, Science and Technology, Republic of Korea.
D
L
D
L
D
D-
D
/L
D/
L
D
L
Although protein structural changes may occur during fluctua-
tions in the environment [24], the active-site residues of related
proteins often retain a very similar geometry despite low the
Appendix A. Supplementary material
overall sequence identity [25e27].
D. turgidum has 61% amino acid sequence identity with that from
L
-Fucose isomerase from
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.biochi.2012.05.009.

1934
S.-H. Hong et al. / Biochimie 94 (2012) 1926e1934
[16] G.D. Haki, S.K. Rakshit, Developments in industrially important thermostable
enzymes: a review, Bioresour. Technol. 89 (2003) 17e34.
[17] J.R. Boulter, W.O. Gielow, Properties of -arabinose isomerase purified from
References
D
[1] C.J. Lin, W.C. Tseng, T.Y. Fang, Characterization of a thermophilic L-rhamnose
two strains of Escherichia coli, J. Bacteriol. 113 (1973) 687e696.
[18] D.A. Case, T.A. Darden, T.E. Cheatham III, C.L. Simmerling, J. Wang, R.E. Duke,
R. Luo, R.C. Walker, W. Zhang, K.M. Merz, B. Roberts, B. Wang, S. Hayik,
A. Roitberg, G. Seabra, I. Kolossváry, K.F. Wong, F. Paesani, J. Vanicek, J. Liu,
X. Wu, S.R. Brozell, T. Steinbrecher, H. Gohlke, Q. Cai, X. Ye, J. Wang, M.J. Hsieh,
G. Cui, D.R. Roe, D.H. Mathews, M.G. Seetin, C. Sagui, V. Babin, T. Luchko,
S. Gusarov, A. Kovalenko, P.A. Kollman, AMBER 11, University of California,
San Francisco, 2010.
isomerase from Caldicellulosiruptor saccharolyticus ATCC 43494, J. Agric. Food
Chem. 59 (2011) 8702e8708.
[2] K. Leang, G. Takada, A. Ishimura, M. Okita, K. Izumori, Cloning, nucleotide
sequence, and overexpression of the L-rhamnose isomerase gene from Pseu-
domonas stutzeri in Escherichia coli, Appl. Environ. Microbiol. 70 (2004)
3298e3304.
[3] C.S. Park, S.J. Yeom, Y.R. Lim, Y.S. Kim, D.K. Oh, Characterization of
a recombinant thermostable L-rhamnose isomerase from Thermotoga mar-
[19] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb,
J.R. Cheeseman, J.J.A. Montgomery, T. Vreven, K.N. Kudin, J.C. Burant,
J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi,
G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara,
K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian, J.B. Cross, C. Adamo,
J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi,
C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador,
J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain,
O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz,
Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko,
P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng,
A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen,
M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian 03, Revision C.02, Gaussian Inc,
Wallingford CT, 2004.
[20] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, AutoDock4 and AutoDockTools4: automated docking with selective
receptor flexibility, J. Comput. Chem. 30 (2009) 2785e2791.
[21] M.M. Bradford, A rapid and sensitive method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein-dye binding, Anal.
Biochem. 72 (1976) 248e254.
[22] M.R. Wilkins, E. Gasteiger, A. Bairoch, J.C. Sanchez, K.L. Williams, R.D. Appel,
D.F. Hochstrasser, Protein identification and analysis tools in the ExPASy
server, Methods Mol. Biol. 112 (1999) 531e552.
[23] K. Guruprasad, B.V. Reddy, M.W. Pandit, Correlation between stability of
a protein and its dipeptide composition: a novel approach for predicting
in vivo stability of a protein from its primary sequence, Protein Eng. 4 (1990)
155e161.
itima ATCC 43589 and its application in the production of
mannose, Biotechnol. Lett. 32 (2010) 1947e1953.
[4] K. Yamanaka, K. Izumori, D-Arabinose (L-fucose) isomerase from Aerobacter
aerogenes, Methods Enzymol. 41 (1975) 462e465.
[5] M. Rhimi, S. Bejar, Cloning, purification and biochemical characterization of
L-lyxose and L-
metallic-ions independent and thermoactive L-arabinose isomerase from the
Bacillus stearothermophilus US100 strain, Biochim. Biophys. Acta 1760 (2006)
191e199.
[6] L. Cheng, W. Mu, T. Zhang, B. Jiang, An L-arabinose isomerase from Acid-
othermus cellulolyticus ATCC 43068: cloning, expression, purification, and
characterization, Appl. Microbiol. Biotechnol. 86 (2010) 1089e1097.
[7] D. Brat, E. Boles, B. Wiedemann, Functional expression of a bacterial xylose
isomerase in Saccharomyces cerevisiae, Appl. Environ. Microbiol. 75 (2009)
2304e2311.
[8] R.D. Whitaker, Y. Cho, J. Cha, H.L. Carrell, J.P. Glusker, P.A. Karplus, C.A. Batt,
Probing the roles of active site residues in D-xylose isomerase, J. Biol. Chem.
270 (1995) 22895e22906.
[9] A. Haldrup, S.G. Petersen, F.T. Okkels, The xylose isomerase gene from Ther-
moanaerobacterium thermosulfurogenes allows effective selection of transgenic
plant cells using D-xylose as the selection agent, Plant Mol. Biol. 37 (1998)
287e296.
[10] J.E. Seemann, G.E. Schulz, Structure and mechanism of L-fucose isomerase
from Escherichia coli, J. Mol. Biol. 273 (1997) 256e268.
[11] E. Garcia-Junceda, G.J. Shen, R. Alajarin, C.H. Wong, Cloning and over-
expression of rhamnose isomerase and fucose isomerase, Bioorg. Med. Chem.
3 (1995) 1349e1355.
[12] Y.H. Ju, D.K. Oh, Characterization of a recombinant
L-fucose isomerase from
Caldicellulosiruptor saccharolyticus that isomerizes
L
-fucose, -arabinose, D-
D
[24] A.M. Lesk, C. Chothia, How different amino acid sequences determine similar
protein structures: the structure and evolutionary dynamics of the globins,
J. Mol. Biol. 136 (1980) 225e230.
[25] A.M. Lesk, C. Chothia, Evolution of proteins formed by beta-sheets. II. The core
of the immunoglobulin domains, J. Mol. Biol. 160 (1982) 325e342.
[26] R.J. Read, G.D. Brayer, L. Jurasek, M.N. James, Critical evaluation of comparative
model building of Streptomyces griseus trypsin, Biochemistry 23 (1984)
6570e6575.
altrose, and
[13] K. Takeda, H. Yoshida, K. Izumori, S. Kamitori, X-ray structures of Bacillus
pallidus -arabinose isomerase and its complex with -fucitol, Biochim. Bio-
phys. Acta 1804 (2010) 1359e1368.
[14] B.T. Menavuvu, W. Poonperm, K. Takeda, K. Morimoto, T.B. Granstrom,
G. Takada, K. Izumori, Novel substrate specificity of -arabinose isomerase
from Klebsiella pneumoniae and its application to production of -altrose from
-psicose, J. Biosci. Bioeng. 102 (2006) 436e441.
[15] K. Izumori, K. Yamanaka, Activation and inhibition of
Klebsiella aerogenes by -histidine, Agric. Biol. Chem. 43 (1979) 1993e1994.
L-galactose, Biotechnol. Lett. 32 (2009) 299e304.
D
L
D
D
D
[27] J.A. Irving, J.C. Whisstock, A.M. Lesk, Protein structural alignments and func-
tional genomics, Proteins 42 (2001) 378e382.
D
-arabinose isomerase of
L