Bivalent cations and amino-acid composition contribute to the
thermostability of
Bacillus licheniformis
xylose isomerase
Claire Vieille
1
, Kevin L. Epting
2
, Robert M. Kelly
2
and J. Gregory Zeikus
1
1
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA;
2
Department of Chemical
Engineering, North Carolina State University, Raleigh, NC, USA
Comparative analysis of genome sequence data from
mesophilic and hyperthermophilic micro-organisms has
revealed a strong bias against specific thermolabile amino-
acid residues (i.e. N and Q) in hyperthermophilic proteins.
The N þ Q content of class II xylose isomerases (XIs)
from mesophiles, moderate thermophiles, and hyperther-
mophiles was examined. It was found to correlate
inversely with the growth temperature of the source
organism in all cases examined, except for the previously
uncharacterized XI from Bacillus licheniformis DSM13
(BLXI), which had an N þ Q content comparable to that
of homologs from much more thermophilic sources. To
determine whether BLXI behaves as a thermostable
enzyme, it was expressed in Escherichia coli, and the

(Co
2þ
–enzyme). These results
suggest that the first irreversible event in BLXI unfolding is
the release of one or both of its metals from the active site.
Although N þ Q content was an indicator of thermo-
stability for class II XIs, this pattern may not hold for other
sets of homologous enzymes. In fact, the extremely
thermostable a-amylase from B. licheniformis was found
to have an average N þ Q content compared with
homologous enzymes from a variety of mesophilic and
thermophilic sources. Thus, it would appear that protein
thermostability is a function of more complex molecular
determinants than amino-acid content alone.
Keywords: Bacillus licheniformis; metal binding; thermo-
stability; xylose isomerase.
It has become apparent that protein thermostability arises
not from a single chemical or physical factor, but from
numerous subtle contributions integrated over the entire
molecular structure [1 – 6]. Thermostable proteins usually
exhibit no significant differences in backbone conformation
when compared with less thermostable proteins, but they
typically have increased numbers of salt bridges, side
chain–side chain hydrogen bonds, and residues involved in
a helices [7–9]. Stability at very high temperatures further
requires that a particular enzyme resist thermally induced
deleterious chemical reactions, which usually occur at
insignificant rates at lower temperatures [10]. For example,
one of the most evident patterns in the amino-acid
composition of hyperthermophilic proteins is the bias

(Received 24 April 2001, revised 29 September 2001, accepted
10 October 2001)
Abbreviations: XI, xylose isomerase; BLXI, Bacillus licheniformis
xylose isomerase; DSC, differential scanning calorimetry.
Eur. J. Biochem. 268, 6291–6301 (2001) q FEBS 2001
Table 1. Amino-acid content of the total proteins of selected mesophiles and hyperthermophiles. Calculations were performed using all the open reading frames described in the genomic sequences present in
GenBank.
Organism Ala Cys Asp Glu Phe Gly His Ile Lys Leu Met Asn Pro Gln Arg Ser Thr Val Trp Tyr
Hyperthermophiles
Aquifex aeolicus 5.90 0.79 4.32 9.63 5.13 6.75 1.54 7.32 9.40 10.57 1.92 3.60 4.07 2.04 4.91 4.79 4.21 7.93 0.93 4.13
Archaeoglobus fulgidus
a
7.84 1.17 4.89 8.90 4.59 7.26 1.51 7.25 6.86 9.49 2.62 3.23 3.86 1.78 5.79 5.51 4.16 8.60 1.04 3.64
Aeropyrum pernix
a
9.53 0.93 3.88 6.61 2.75 8.55 1.92 5.19 3.52 11.38 1.97 2.04 6.46 1.90 7.71 7.53 4.69 8.75 1.31 3.36
Methanococcus jannaschii
a
5.54 1.27 5.52 8.67 4.20 6.41 1.43 10.45 10.36 9.38 2.33 5.24 3.38 1.44 3.85 4.46 4.06 6.85 0.71 4.33
Pyrococcus abyssi
b
6.68 0.55 4.61 8.85 4.35 7.26 1.50 8.50 7.80 10.25 2.40 3.34 4.26 1.67 5.73 4.97 4.20 8.07 1.18 3.83
Pyrococcus horikoshii
b
6.37 0.63 4.26 8.29 4.60 6.97 1.49 8.79 7.74 10.36 2.40 3.54 4.51 1.63 5.46 5.86 4.51 7.55 1.17 3.84
Thermotoga maritima 5.85 0.71 4.96 8.92 5.19 6.92 1.58 7.22 7.61 10.02 2.40 3.63 3.99 2.01 5.55 5.65 4.52 8.60 1.10 3.58
Average 6.82 0.86 4.63 8.55 4.40 7.16 1.57 7.82 7.61 10.21 2.29 3.52 4.36 1.78 5.57 5.54 4.34 8.05 1.06 3.82
Variance 1.72 0.06 0.25 0.77 0.57 0.4 0.02 2.31 4.02 0.4 0.06 0.75 0.84 0.04 1.15 0.88 0.05 0.4 0.03 0.09
Standard deviation 1.31 0.25 0.5 0.88 0.76 0.63 0.15 1.52 2.00 0.63 0.23 0.87 0.92 0.20 1.07 0.94 0.22 0.63 0.18 0.31
Mesophiles

mation is available [10,17–24]. On the basis of the absence
or presence of a 50-residue insert at the N-terminus, XIs
have been classified into class I and class II enzymes,
respectively [25]. Two distinct metal-binding sites, M1 and
M2, have been identified in all XIs: (a) the metal in site M1
is co-ordinated to four carboxylate groups; (b) the metal in
site M2 is co-ordinated to one imidazole and three
carboxylate groups. The metals in sites M1 and M2 were
initially referred to as structural and catalytic metals,
respectively [18,26,27], but these appellations are no longer
valid, because later studies showed that both metals are
directly involved in catalysis [24,28,29]. The stabilizing and
activating metals are typically the bivalent cations Mg
2þ
,
Co
2þ
, and Mn
2þ
. Metal specificity depends on both the
nature of the substrate (i.e. glucose or xylose) and whether
the enzyme is a class I or class II XI. Thermus aquaticus XI,
a class I enzyme, isomerizes glucose most efficiently
when in the presence of Mn
2þ
, but its activity toward
xylose is highest with Co
2þ
as the cofactor [26]. The class II
Bacillus coagulans XI, on the other hand, isomerizes

not available to make this determination. In an attempt to
test the simple hypothesis that class II XI stability at high
temperatures correlates with its N þ Q content, independent
of the growth temperature of the source organism, the
biochemical and biophysical properties of the previously
uncharacterized BLXI were determined. Particular empha-
sis was placed on the influence of bivalent cations on activity
and stability. Our results show that for class II XIs, N þ Q
content relates to the enzyme’s functional temperature range
and that BLXI thermostability is also directly related to the
binding of specific metals as cofactors. At the same time, the
simple relationship between thermostability and N þ Q
content may not hold in general, as it is not the case for the
thermostable a-amylase from B. licheniformis.
MATERIALS AND METHODS
B. licheniformis xylA
gene cloning
B. licheniformis strain DSM13 was grown at 37 8Cin
Luria–Bertani broth [31]. A B. licheniformis genomic DNA
library was constructed in vector pUC18 (Pharmacia,
Piscataway, NJ, USA), using methods described in [23].
E. coli xyl
–
mutant HB101 (F
–
, hsdS20, ara-1, recA13,
proA12, lacY1, galK2, rpsL20, mtl-1, xyl-5 ) [32] was
transformed with the ligation mixture by electroporation and
plated on M9 medium containing 0.2% xylose, 0.1%
casamino acids, thiamine (500 mg·mL

Coomassie blue R250. The homogeneous fractions were
pooled and extensively dialyzed against buffer A. Protein
concentrations were determined using the Bio-Rad protein
assay kit (Bio-Rad, Richmond, CA, USA), with BSA as the
standard. The purified enzyme was stored at 2 70 8C until
use.
Molecular mass determination
BLXI molecular mass was determined by gel filtration using
a Sephacryl S-300 HR column (1.4 cm £ 160 cm)
calibrated with blue dextran and protein standards of 443,
200, 150, and 66 kDa (Sigma Chemical Co., St Louis, MO,
USA). The flow rate was 0.2 mL·min
21
.
EDTA treatment
The purified enzyme was incubated overnight at 4 8Cin
buffer A containing 10 m
M EDTA. It was then dialyzed
twice against 50 m
M Mops (pH 7.0) (SigmaUltra, Sigma
Chemical Co.) containing 2 m
M EDTA, and finally dialyzed
twice against 50 m
M Mops (pH 7.0), this time without
EDTA. The apoenzyme was divided into aliquots and stored
at 2 70 8C until use.
Enzyme assays
BLXI activity was assayed routinely with glucose as the
substrate. The enzyme (0.06 mg·mL
21

, or MgCl
2
were added to the apoenzyme
reaction mixture at concentrations of 0.003– 100 m
M
(activity on glucose) or 0.002– 0.03 mM (activity on
xylose). Activity on xylose was determined using
0.024 mg·mL
21
apo-BLXI and 635 mM xylose. Activity
on glucose was determined using 0.06 mg·mL
21
apo-BLXI
and 1
M glucose. The activation constant, K
act
, for a metal is
defined as the metal concentration that results in 50% of
maximum activity [34,35].
Enzyme thermoinactivation
The apo-BLXI (0.17 mg·mL
21
)in50mM Mops (pH 7.0 at
room temperature) was incubated at various temperatures
(in a Perkin –Elmer Cetus GeneAmp PCR system 9600) for
different periods of time. Thermoinactivation was stopped
by transferring the tubes to an ice bath. Residual activity was
determined under the conditions described above, except
that the reaction mixture contained 5 m
M CoCl

cuvette-heating system. The increasing thermal gradient
was 1.0 8C·min
21
. The effect of metals on apo-BLXI
precipitation was studied in the presence of 0.5 m
M CoCl
2
,
0.5 m
M MnCl
2
,or5mM MgCl
2
.
PH studies
The effect of pH on BLXI activity was determined at 64 8C
using the routine assay described above, except that the
Mops buffer was substituted with 100 m
M sodium acetate
(pH 4.0–5.7), 100 m
M Pipes (pH 6.0–7.5), or 100 mM
Hepps (pH 7.5–8.7). All pHs were adjusted at room
temperature, and the DpK
a
/DT values for acetate, Pipes, and
Hepes (0.000, 2 0.0085, and 2 0.011, respectively) [36]
were taken into account for the results.
Differential scanning calorimetry (DSC)
DSC experiments were performed on a Nano-Cal differen-
tial scanning calorimeter (Calorimetry Sciences Corp.,

Fig. 1. Determination of BLXI molecular mass by gel filtration.
V
e
/V
o
is the ratio of a protein’s elution volume to the elution volume of
blue dextran. (X) Protein standards; (A) BLXI. Linear regression (1),
with an r
2
of 0.951, is based on the elution data of all four protein
standards. Linear regression (2), with an r
2
of 0.981, is based on the
elution data of the 200, 150, and 66 kDa protein standards. Linear
regressions (1) and (2) give molecular masses of 200 and 177.5 kDa,
respectively, for BLXI.
6294 C. Vieille et al.(Eur. J. Biochem. 268) q FEBS 2001
that pBL1 contained B. licheniformis xylR, xylA, and a
truncated xylB. A 1.2-kb Sph I–Eco RI fragment was deleted
from the pBL1 insert to inactivate the B. licheniformis xylR
repressor gene, leading to plasmid pBL2. Plasmid pBL2 was
used to express BLXI in the rest of the study.
Purification of the recombinant protein and physical
properties
The B. licheniformis xylA gene was expressed from its own
promoter in plasmid pBL2. The recombinant enzyme was
purified (heat treatment plus DEAE–Sepharose chromato-
graphy) from an HB101 (pBL2) 2-L culture grown in M9
medium plus xylose. The purified enzyme was shown to be
homogeneous by SDS/PAGE and staining with Coomassie

activities (70 and 80 kJ·mol
21
, respectively; unpublished
data).
The effect of pH on BLXI activity was determined by
measuring the holoenzyme activity on glucose between
pH 4.8 and 8.2 (values after temperature correction). BLXI
is optimally active at pH 7.2 and shows more than 80%
activity between pH 6.8 and 7.6 (Fig. 2B).
BLXI kinetic parameters
BLXI kinetic parameters were determined at 60 8C for
glucose and xylose using the holoenzyme in the presence of
1m
M CoCl
2
(Table 2). Not surprisingly, BLXI is about six
times more efficient on xylose than on glucose, as indicated
by the values of V
max
/K
m
. Compared with other type II
thermophilic XIs (Table 2), BLXI has average kinetic
parameters, but it has a relatively low catalytic efficiency on
xylose.
BLXI thermostability and inactivation characteristics
BLXI thermostability was first characterized using the
holoenzyme in the presence of 1 m
M CoCl
2

K
m
(mM)
B. licheniformis DSM13 60 7.7 145 0.053 22.2 67 0.33 This work
B. stearothermophilus 60 6.0 220 0.027 44.5 100 0.44 [55]
T. saccharolyticum B6A 65 6.3 120 0.053 17.6 16 1.10 [56]
T. thermosulfurigenes 4B 65 5.3 142 0.037 15.7 20 0.78 [56]
T. maritima DSM 3109 90 16.2 118 0.137 68.4 74 0.92 [57]
T. neapolitana 5068 90 22.4 89 0.253 52.2 16 3.28 [23]
Fig. 2. Effects of temperature and pH on BLXI
specific activity. (A) Arrhenius plot of BLXI
specific activity as a function of temperature. The
linear regression was only applied to the
temperature points below the optimum
temperature for activity. (B) Effect of pH on BLXI
activity. Assays were performed at 64 8Cin
100 m
M sodium acetate (A; pH 4.0– 5.7), 100 mM
Pipes (X; pH 6.0 –7.5), or 100 mM Hepes (K
pH 7.5 –8.7). The DpK
a
/DT values for acetate,
Pipes, and Hepps were taken into account to
calculate the pH values at 64 8C. All assays were
performed in triplicate.
q FEBS 2001 B. licheniformis xylose isomerase (Eur. J. Biochem. 268) 6295
(Fig. 3A; 0.012, 0.01, and 0.007 min
21
at 0.05, 0.5, and
2.5 mg·mL

Metal requirement for BLXI activity
To determine which metal cation (Co
2þ
,Mn
2þ
,orMg
2þ
)
best activates BLXI on glucose and on xylose, the
apoenzyme activity was tested on both sugars in the
presence of increasing concentrations of each cation (in
the chloride form; Fig. 4). In the absence of metal, the
apoenzyme was completely inactive on both substrates.
Co
2þ
was by far the best activating cation for BLXI activity
on glucose. BLXI activity in the presence of Mn
2þ
and
Mg
2þ
reached only 6% and 15%, respectively, of the activity
in the presence of Co
2þ
(Fig. 4A). Co
2þ
,Mn
2þ
, and Mg
2þ

Clark [27] obtained a similar result with B. coagulans XI:
K
act
values were 10 times higher for glucose than for xylose,
and the relative effectiveness of the three metals was the
same. Occupancy rate of the M2 site necessary for activity
on glucose may be higher than that for activity on xylose; it
has been found that some XIs are active on xylose with only
the M1 site occupied [27]. Also, the metal-specific
Fig. 3. Characteristics of holo-BLXI inactivation at 68 8C. Assays
were performed in triplicate. All linear regression had correlation
coefficients r
2
above 0.96. (A) Effect of enzyme concentration on holo-
BLXI inactivation rate. Enzyme concentrations: (A) 0.05 mg·mL
21
;
(X) 0.5 mg·mL
21
;(K) 2.5 mg·mL
21
. Inactivation rates corresponding
to the slopes of the linear regressions for the three inactivation curves
were 0.012 min
21
at 0.05 mg·mL
21
, 0.01 min
21
at 0.5 mg·mL

thermostability, the apoenzyme was incubated in the
presence of 0.5 m
M Co
2þ
, 0.5 mM Mn
2þ
,or2mM Mg
2þ
at different temperatures and for various periods of time.
The metal was allowed to equilibrate between the buffer and
the enzyme by preincubating the enzyme–metal mixture at
30 8C for 30 min. Remaining activity was measured with
glucose as the substrate in the presence of 4 m
M CoCl
2
. The
apoenzyme was significantly less stable than the enzyme
containing Co
2þ
,Mn
2þ
,orMg
2þ
(Fig. 5 and Table 3). Of
the three metal cations, Mn
2þ
stabilized BLXI best. Co
2þ
was only slightly less stabilizing than Mn
2þ

from the apoenzyme to the Mn
2þ
-containing enzyme, loss
of the metal cofactor could be the limiting step in BLXI
inactivation. Higher E
a
values of inactivation for the Co
2þ
–
enzyme and the Mn
2þ
–enzyme reflect the higher thermal
energy that these metal–enzyme complexes can accumulate
before losing the tightly bound metal, causing them to
unfold. The higher E
a
of inactivation provided by Co
2þ
and
Mn
2þ
compared with that provided by Mg
2þ
reflects the
different binding affinities of these cations for BLXI.
The extremely high E
a
of BLXI inactivation in the
presence of Co
2þ

(X).
Table 3. Effect of metals on apo-BLXI stability. NI, Data not interpretable.
Metal
Kinetic stability
Thermodynamic stability:
Half-life
(min)
E
a
of
inactivation
(kJ·mol
21
)
Precipitation
temperature
(8C)
Melting
temperature
(8C)
No metal 24 (at 40 8C) 342 NI 50.3
2m
M MgCl
2
53 (at 54 8C) 604 57.1 53.3
35 (at 56 8C)
0.5 m
M CoCl
2
53 (at 69.5 8C) 1166 69.9 73.4

precipitation temperature. This temperature increased in the
order Mg
2þ
–BLXI , Co
2þ
–BLXI , Mn
2þ
–BLXI.
The precipitation temperatures in the presence of metals
correlate well with the inactivation data (Table 3).
Precipitation experiments with the apoenzyme did not
provide reproducible data (not shown).
The melting temperature for BLXI was determined by
DSC in the presence and absence of metals (Fig. 7 and
Table 3). It increased in the order apo-BLXI , Mg
2þ
–
BLXI , Co
2þ
–BLXI , Mn
2þ
–BLXI , (Mg
2þ
þ
Co
2þ
)–BLXI. These values are consistent with BLXI
inactivation and precipitation temperatures.
The lower stability and lower E
a

Mn
2þ
[41]. Whereas Co
2þ
and Mn
2þ
are present in both
metal sites in crystals of Arthrobacter XI at pH 6.0, crystals
of Arthrobacter XI do not contain any Mg
2þ
at pH 6.0, and
contain Mg
2þ
only in site M1 at pH 8.0 [42]. As all
Fig. 7. Thermal unfolding of apo-BLXI in the presence and
absence of metals followed by DSC. See Materials and methods for
experimental details.
Fig. 8. Distribution of the N 1 Q (A), Q (B),
and N (C) contents in the 90 B. licheniformis
proteins of known sequence. Sequences were
obtained from GenBank.
6298 C. Vieille et al.(Eur. J. Biochem. 268) q FEBS 2001
inactivation experiments in this work were performed at
pH 7.0, Mg
2þ
binding was probably not optimal. The
difference in BLXI stabilization provided by Mg
2þ
v Co
2þ

[45,46], whereas Mn
2þ
is hexa-co-ordinated and adopts an
octahedral geometry [18].
N 1 Q content as a general indicator of protein
thermostability in mesophiles
We reported previously [23] that the N þ Q content of class
II XIs correlated with the growth temperature of the source
organism, with the notable exception of BLXI. It is also
interesting that of the B. licheniformis proteins with
sequence available, the Q and N þ Q contents in BLXI
are among the lowest (Fig. 8A,B). This is not the case,
however, for the N content of BLXI (Fig. 8C). The entire
genome of several mesophilic and hyperthermophilic
organisms were analyzed in recent studies [9,47–49]. All
these studies reported a decrease in the content of uncharged
polar amino acids (i.e. Q, N, S, and T) and an increase in
charged amino-acid residues (i.e. K, E, and R) in
hyperthermophilic proteins. As S and T can catalyze the
deamination and backbone cleavage of Q and N residues
[48,50], a reduction in all four of these residues would
minimize deamination.
Although our prediction that BLXI, based on its low
N þ Q content, had thermophilic properties (i.e. high
thermostability and optimal activity at high temperatures)
proved to be correct, a high N þ Q content does not
necessarily predict that an enzyme will be thermolabile.
This observation is evident from Fig. 8: the B. licheniformis
a-amylase, which has an N þ Q content higher than the
average (4.88% N, 4.30% Q), is an extremely thermostable

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