The energy of activation for irreversible inactivation was also strongly influenced by the metal present, ranging from suggest that the first irreversible event in BLXI unfolding is the
Trang 1Bivalent cations and amino-acid composition contribute to the
1 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA;2Department 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
thermostability and activity properties of the recombinant
enzyme were studied Indeed, it was optimally active at
70 – 72 8C, which is significantly higher than the optimal
growth temperature (37 8C) of B licheniformis The
kinetic properties of BLXI, determined at 60 8C with
glucose and xylose as substrates, were comparable to
those of other class II XIs The stability of BLXI was
dependent on the metallic cation present in its two
tempera-tures of 50.3 8C, 53.3 8C, 73.4 8C, and 73.6 8C BLXI inactivation was first-order in all conditions examined The energy of activation for irreversible inactivation was also strongly influenced by the metal present, ranging from
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
against thermally labile amino-acid residues This pattern is obvious on examination of the amino-acid compositions of the total protein content of eight mesophilic and seven hyperthermophilic micro-organisms for which genome sequence data are available (Table 1) The most striking difference is the 55% reduction in the number of glutamines
in hyperthermophilic proteins; note also the 28% reduction
in asparagines As these two amino acids are easily deamidated at elevated temperatures [10 – 13], it is not surprising that they are less abundant in proteins from hyperthermophiles This observation raises the question of whether a relatively low N þ Q content is a signature of enhanced thermostability in proteins from mesophilic sources
The potential use of biocatalysts at high temperatures for technological purposes has drawn interest in developing thermoactive and thermostable enzymes that would provide significant processing advantages [14] For example, thermostable xylose isomerases (XIs) (EC 5.3.1.5), which
the production of high-fructose corn syrup [15] Elevated bioprocessing temperatures are preferred to achieve higher catalytic rates as well as more favorable equilibrium yields
Correspondence to J G Zeikus, Department of Biochemistry and
Molecular Biology, 410 Biochemistry Building, Michigan State
University, East Lansing, MI 48824, USA Fax: þ 517 353 9334,
Tel.: þ 517 353 5556, E-mail: zeikus@msu.edu
(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.
Trang 22.16) 1%
b Archaea
Trang 3[16] Because of the commercial importance of XIs, their
biochemical, biophysical, and structural properties have
been extensively studied, and abundant sequence
infor-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
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
Bacillus coagulans XI, on the other hand, isomerizes
whereas its activity toward fructose is best promoted by
Class I XIs are a relatively homogeneous group when it
comes to thermostability, whereas class II XIs vary widely
in this regard This heterogeneity among class II XIs may
arise from the existence of additional salt bridge(s) specific
to thermostable class II XIs [30] In a previous study of class
II XIs, a positive correlation between the enzyme’s N þ Q
content and the growth temperature of the source organism
was observed: XIs from the most thermophilic sources
typically had lower N þ Q content [23] Of the XIs
examined, only the enzyme from the mesophilic bacterium
Bacillus licheniformis DSM13 (BLXI) was atypical
Originating from an organism that optimally grows at
37 8C, BLXI contains only 26 N þ Q residues, compared
with 23 in the enzyme from the hyperthermophile
Thermotoga neapolitana (optimal growth at 80 8C), and
46 in the XI from the mesophile Escherichia coli (optimal
growth at 37 8C) The low Q (and, to some extent, N)
content in proteins from hyperthermophiles (Table 1) raises
an interesting question: does a relatively low N þ Q content
in a protein from a mesophile indicate an unusually high
thermostability for this protein? The location of N and Q
residues within a protein structure is certainly a critical
consideration, but detailed structural information is often
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
M A T E R I A L S A N D M E T H O D S
B licheniformis xylA gene cloning
B licheniformis strain DSM13 was grown at 37 8C in Luria – Bertani broth [31] A B licheniformis genomic DNA library was constructed in vector pUC18 (Pharmacia, Piscataway, NJ, USA), using methods described in [23]
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%
recombinant XI produced large colonies on this medium Enzyme purification
BLXI was purified from a 2-L culture of E coli HB101 carrying plasmid pBL2 grown in M9 medium (comple-mented as above) After centrifugation for 5 min at 4000 g,
cells were disrupted by two consecutive passes through a French pressure cell (American Instrument Co., Silver Spring, MD, USA), using a decrease in pressure of 96.5 MPa After centrifugation at 25 000 g for 30 min, the supernatant was heat-treated at 60 8C for 10 min The precipitated material was separated by centrifugation at
25 000 g for 30 min The soluble fraction was loaded on to
a DEAE – Sepharose Fast-Flow column equilibrated with
NaCl gradient in buffer A The active fractions were analyzed by SDS/PAGE (12% acrylamide) and stained with 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,
EDTA treatment The purified enzyme was incubated overnight at 4 8C in
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
Trang 450 mM Mops (pH 7.0 at room temperature) containing
reaction was stopped by transferring the tubes to an ice bath
The amount of fructose produced was determined by the
cysteine/carbazole/sulfuric acid method [33] To determine
the effect of temperature on BLXI activity, the holo-BLXI
was incubated in the reaction mixture at the temperatures of
interest in a Perkin – Elmer Cetus GeneAmp PCR system
9600 (Perkin – Elmer, Norwalk, CT, USA) for 20 min To
determine the kinetic parameters, assays were performed in
xylose The amounts of fructose and xylulose produced were
determined using the cysteine/carbazole/sulfuric acid
method Absorbance was measured at 537 nm and 560 nm
for xylulose and fructose, respectively One unit of
isomerase activity is defined as the amount of enzyme that
produces 1 mmol of product per min under the assay
conditions
Activation of apo-BLXI by metals
To examine the effect of bivalent cations on enzyme activity,
xylose) Activity on xylose was determined using
defined as the metal concentration that results in 50% of
maximum activity [34,35]
Enzyme thermoinactivation
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
resulting solution was equilibrated for 30 min at 30 8C
before thermoinactivation was initiated (preincubation
conditions known to be sufficient for the metal to reach
equilibrium between the buffer and enzyme metal-binding
sites [19])
Heat-induced enzyme precipitation
Heat-induced enzyme precipitation was monitored from
25 8C to 90 8C by light scattering (l ¼ 580 nm), using the
Absorbance measurements were conducted in 0.3-mL
quartz cuvettes (pathlength 1 cm) using a Beckman
DU-650 spectrophotometer equipped with a Peltier
cuvette-heating system The increasing thermal gradient
PH studies The effect of pH on BLXI activity was determined at 64 8C using the routine assay described above, except that the
Hepps (pH 7.5 – 8.7) All pHs were adjusted at room
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.,
were scanned from 25 8C to 100 8C The apoenzyme (obtained by EDTA treatment, see above) was scanned
enzymes, the apoenzyme was incubated for 2 h at room
to remove the metal that was not tightly bound to the enzyme Each metal-containing enzyme was scanned against the corresponding dialysis buffer The dialysis buffer was used to generate the baseline The enzyme
A, then scanned against the dialysis buffer as control
R E S U L T S A N D D I S C U S S I O N
Plasmid pBL1 was characterized from an HB101 transfor-mant that formed large colonies on M9 medium containing xylose Comparison of the physical map of the plasmid pBL1 insert with that of plasmid pWH1450 [37] indicated
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 r2of 0.951, is based on the elution data of all four protein standards Linear regression (2), with an r2of 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.
Trang 5that 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
blue Approximately 100 mg enzyme was obtained from the
2-L culture A molecular mass of 200 kDa for the native
protein was estimated by gel filtration (Fig 1) Analysis by
SDS/PAGE showed a single band with a molecular mass of
about 50 kDa This estimate is in agreement with that
expected from the protein sequence (50 905 Da) These
results indicate that BLXI is expressed as a homotetramer
in E coli It is interesting to note that XIs from the
thermophile Thermoanaerobacterium thermosulfurigenes
and the hyperthermophile T neapolitana, both
homotetra-mers in their native forms, are expressed as active dihomotetra-mers
in E coli [38]
Effects of temperature and pH on BLXI activity
The effect of temperature on BLXI activity was determined
by measuring the holoenzyme activity on glucose in the
optimally active between 70 8C and 72 8C Above 72 8C, the enzyme rapidly loses activity, and it is completely inactive at
80 8C The Arrhenius plot for BLXI activity is linear between 35 8C and 66 8C The estimated energy of
value comparable to those for E coli and T neapolitana XI
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
times more efficient on xylose than on glucose, as indicated
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
conditions, BLXI has a half-life of 14 h at 64 8C, 70 min at
67 8C, and 2 min 40 s at 70 8C Figure 3 shows that BLXI inactivation at 68 8C was first-order Inactivation rates obtained for three different enzyme concentrations at 68 8C
Table 2 Kinetic constants of thermophilic type II XIs.
Organism
T (8C)
Reference
V max
(U·mg 21 )
K m
(m M ) V max /K m
V max
(U·mg 21 ) V max /K m
K m
(m M )
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 8C in
100 m M sodium acetate (A; pH 4.0–5.7), 100 m M
Pipes (X; pH 6.0–7.5), or 100 m M 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.
Trang 6(Fig 3A; 0.012, 0.01, and 0.007 min21 at 0.05, 0.5, and
inactivation is slightly dependent on concentration, with
BLXI stability increasing with enzyme concentration At 0.5
BLXI inactivation Residual activities were compared in
whole-enzyme (sample before centrifugation) and soluble
(supernatant after centrifugation) fractions after BLXI
inactivation at 68 8C The soluble fraction showed an
inactivation rate that was slightly higher than the whole
enzyme (Fig 3B) This difference does not appear to be
significant Inactivation was accompanied by heavy
aggregation at this enzyme concentration, and the difference
in inactivation rate between the two samples probably
results from the trapping of some active soluble enzyme
molecules in the aggregate As the aggregate increased in size with inactivation time, more and more soluble enzyme may remain trapped in the insoluble pellet during centrifugation These results suggest that the precipitated enzyme was completely inactive, and that the soluble fraction remained fully active The Arrhenius plot of
inactivation mechanism that involves significant unfolding [10]
Metal requirement for BLXI activity
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
activation constants for BLXI activity on glucose are
The metal requirement of BLXI for activity on xylose was significantly different from its metal requirement for activity
on glucose The three metals studied stimulate BLXI
times lower than for BLXI activity on glucose Marg & Clark [27] obtained a similar result with B coagulans XI:
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 r2above 0.96 (A) Effect of enzyme concentration on
holo-BLXI inactivation rate Enzyme concentrations: (A) 0.05 mg·mL 21 ;
(X) 0.5 mg·mL21; (K) 2.5 mg·mL21 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 21 , and
0.007 min21at 2.5 mg·mL21 (B) Remaining activity in the total and
soluble holo-BLXI fractions Initial enzyme concentration was
2.5 mg·mL 21 The soluble fraction corresponded to the supernatant
of the whole enzyme fraction, after centrifugation (K) Whole enzyme;
(O) soluble fraction.
Fig 4 Apo-BLXI activation by Co 21
(A), Mn 21
(X), and Mg 21
(S) (A) Apo-BLXI specific activity with glucose as the substrate (B) and (C) Apo-BLXI specific activity with xylose as the substrate Assays were performed in triplicate.
Trang 7differences observed in enzyme – metal binding are probably
related to differences between the metals with respect to
co-ordination geometries These differences may influence
the metal preferences for glucose as opposed to xylose
isomerization For example, whereas Mn – BLXI was highly
active on xylose, it was barely active on glucose
Metal requirement of BLXI for stability
To determine the metal requirement of BLXI for
thermostability, the apoenzyme was incubated in the
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
apoenzyme was significantly less stable than the enzyme
metals at stabilizing BLXI This metal-specific protection of BLXI against inactivation is very similar to the situation
the apoenzyme in the presence and absence of metals (Table 3) The nature of the metal present clearly affected
specific activity decreased rapidly above 72 8C (Fig 2) As
enzyme starts inactivating at a measurable rate increase
of the metal cofactor could be the limiting step in BLXI
energy that these metal – enzyme complexes can accumulate before losing the tightly bound metal, causing them to
different binding affinities of these cations for BLXI
Fig 5 Arrhenius plots of apo-BLXI inactivation rates in the
absence (K) or presence of 0.5 m M Co 21
(A), 0.5 m M Mn 21
(X), or
2 m M Mg21(S) All linear regression had correlation coefficients r2
above 0.99.
Fig 6 Determination of apo-BLXI precipitation temperature in the presence of 2 m M Mg21(S), 0.5 m M Co21(A), or 0.5 m M Mn21 (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·mol21)
Precipitation temperature (8C)
Melting temperature (8C)
35 (at 56 8C)
18 (at 70.5 8C)
16 (at 72.5 8C)
Trang 8Co2þ (C Vieille & J G Zeikus, unpublished results).
inactivation for BLXI and T thermosulfurigenes XI are
related to differences in metal co-ordination geometries
Figure 6 and Table 3 show the effect of metals on BLXI precipitation temperature This temperature increased in the
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
inactivation and precipitation temperatures
xylose) However, the influence of pH on the decreased
needs to be considered The metals bind the enzyme through one His imidazole and several carboxylate groups By
metal sites in crystals of Arthrobacter XI at pH 6.0, crystals
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.
Trang 9inactivation experiments in this work were performed at
a higher pH
The significance of cation binding in XI stability has not
yet been examined closely However, some information on
this issue is available Site-directed mutagenesis has been
used to partially fill a metal-binding site with the side chain
of an amino acid These mutations to both metal-binding
sites, M1 and M2, resulted in destabilized XIs For example,
mutation of His220 in the class I S rubiginosus XI affected
metal binding at M2, which in turn was responsible for
destabilization [35] A similar observation has been made
for the class II E coli XI: mutation of His271 (ligand to
metal 2) significantly destabilized the enzyme without
changing its structure appreciably (as determined by CD
analysis) [43] Other point mutations triggering
confor-mational changes in active-site residues have also been
found to destabilize XIs [44], suggesting that thermal
unfolding starts through movements of active-site residues
Metal cations probably act to lock the active site in a stable
conformation, which is lost as soon as the metal leaves the
active site Specific differences in stabilization efficacy
between metals may be due to their ability to adopt different
geometries in the same site in the absence of substrate In the
crystal structures of the S rubiginosus enzyme, for
penta-co-ordinated and it adopts a strongly distorted geometry
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
enzyme, with optimal activity at 90 8C [51] In addition, the
N and Q contents of B licheniformis a-amylase are close to
the average N and Q contents (5.04 ^ 1.25% and
3.77 ^ 1.03%, respectively) of 20 homologous a-amylases
from micro-organisms with differing growth temperature optima (data not shown) It is interesting to note, however, that five out of the seven thermostabilizing mutations that have been identified in this a-amylase (by Declerk et al [52,53]) are substitutions of N or Q residues by less thermolabile amino acids Thus, it appears that the N þ Q content alone does not account for thermostability, and that the location of these residues in the protein’s 3D structure must be taken into account
A C K N O W L E D G E M E N T S
This work was supported by the US National Science Foundation, grants NSF-Bes-9809964 (MSU) and NSF-Bes 9817067 (NCSU) We express our deep gratitude to Dr A Roy Day for his help with the statistical analysis in Table 1 and Christopher B Jambor for editing the manuscript.
R E F E R E N C E S
1 Beadle, B.M., Baase, W.A., Wilson, D.B., Gilkes, N.R & Shoichet, B.K (1999) Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme Biochemistry 38,
2570 – 2576.
2 Hollien, J & Marqusee, S (1999) Structural distribution of stability
in a thermophilic enzyme Proc Natl Acad Sci USA 96, 13674– 13678.
3 Hasegawa, J., Uchiyama, S., Tanimoto, Y., Mizutani, M., Kobayashi, Y., Sambongi, Y & Igarashi, Y (2000) Selected mutations in a mesophilic cytochrome c confer the stability of a thermophilic counterpart J Biol Chem 275, 37824 – 37828.
4 Numata, K., Hayashi-Iwasaki, Y., Kawaguchi, J., Sakurai, M., Moriyama, H., Tanaka, N & Oshima, T (2001) Thermostabiliza-tion of a chimeric enzyme by residue substituThermostabiliza-tions: four amino acid residues in loop regions are responsible for the thermostability of Thermus thermophilus isopropylmalate dehydrogenase Biochim Biophys Acta 1545, 174 – 183.
5 Petsko, G.A (2001) Structural basis of thermostability in hyperthermophilic proteins, or "there’s more than one way to skin a cat" Methods Enzymol 334, 469 – 478.
6 Vieille, C & Zeikus, J.G (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability Microbiol Mol Biol Rev 65, 1 – 43.
7 Adams, M.W & Kelly, R.M (1998) Finding and using hyperthermophilic enzymes Trends Biotechnol 16, 329 – 332.
8 Elcock, A.H (1998) The stability of salt bridges at high temperatures: implications for hyperthermophilic proteins J Mol Biol 284, 489 – 502.
9 Cambillau, C & Claverie, J.-M (2000) Structural and genomic correlates of hyperthermostability J Biol Chem 42, 32383– 32386.
10 Hess, J.M & Kelly, R.M (1999) Influence of polymolecular events
on inactivation behavior of xylose isomerase from Thermotoga neapolitana 5068 Biotechnol Bioeng 62, 509 – 517.
11 Ahern, T.J & Klibanov, A.M (1985) The mechanisms of irreversible enzyme inactivation at 1008C Science 228,
1280 – 1284.
12 Tomazic, S.J & Klibanov, A.M (1988) Mechanisms of irreversible thermal inactivation of Bacillus a-amylases J Biol Chem 263,
3086 – 3091.
13 Tomazic, S.J & Klibanov, A.M (1988) Why is one Bacillus a-amylase more resistant against irreversible thermoinactivation than another? J Biol Chem 263, 3092 – 3096.
14 Adams, M.W.W., Perler, F.B & Kelly, R.M (1995) Extremozymes: expanding the limits of biocatalysis Bio/Technol 13, 662 – 668.
15 Bentley, I.S & Williams, E.C (1996) Starch conversion In
Trang 10Industrial Enzymology (Godfrey, T &West, S.I., eds), pp 339 – 357.
Stockton Press, New York.
16 Converti, A & Del Borghi, M (1998) Kinetics of glucose
isomerization to fructose by immobilized glucose isomerase in the
presence of substrate protection Bioprocess Engineering 18,
27 – 33.
17 Collyer, C.A & Blow, D.M (1990) Observations of reaction
intermediates and the mechanism of aldose-ketose interconversion
by D -xylose isomerase Proc Natl Acad Sci USA 87, 1362 – 1366.
18 Whitlow, M., Howard, A.J., Finzel, B.C., Poulos, T.L., Winborne,
E & Gilliland, G.L (1991) A metal-mediated hydride shift
mechanism for xylose isomerase based on the 1.6 A ˚ Streptomyces
rubiginosus structures with xylitol and D -xylose Proteins 9,
153 – 173.
19 van Bastelaere, P.B., Callens, M., Vangrysperre, W.A &
Kersters-Hilderson, H.L (1992) Binding characteristics of Mn 2þ , Co 2þ and
Mg2þ ions with several D -xylose isomerases Biochem J 286,
729 – 735.
20 van Tilbeurgh, H., Jenkins, J., Chiadmi, M., Janin, J., Wodak, S.J.,
Mrabet, N.T & Lambeir, A.M (1992) Protein engineering of
xylose (glucose) isomerase from Actinoplanes missouriensis 3.
Changing metal specificity and the pH profile by site-directed
mutagenesis Biochemistry 31, 5467 – 5471.
21 Meng, M., Bagdasarian, M & Zeikus, J.G (1993) Thermal
stabilization of xylose isomerase from Thermoanaerobacterium
thermosulfurigenes Bio/Technol 11, 1157 – 1161.
22 Lavie, A., Allen, K.N., Petsko, G.A & Ringe, D (1994) X-ray
crystallographic structures of D -xylose isomerase-substrate
com-plexes position the substrate and provide evidence for metal
movement during catalysis Biochemistry 33, 5469 – 5480.
23 Vieille, C., Hess, J.M., Kelly, R.M & Zeikus, J.G (1995) xylA
cloning and sequencing and biochemical characterization of xylose
isomerase from Thermotoga neapolitana Appl Environ
Micro-biol 61, 1867 – 1875.
24 Bogumil, R., Kappl, R & Hu¨ttermann, J (2000) Role of the
binuclear manganese (II) site in xylose isomerase In Metal Ions in
Biological Systems, vol 37, pp 366 – 405 Marcel Dekker, Inc.,
New York.
25 Vangrysperre, W., Van Damme, J., Vandekerckhove, J., De Bruyne,
C.K., Cornelis, R & Kersters-Hilderson, H (1990) Localization of
the essential histidine and carboxylate group in D -xylose
isomerases Biochem J 265, 699 – 705.
26 Lehmacher, A & Bisswanger, H (1990) Comparative kinetics of
D -xylose and D -glucose isomerase activities of the D -xylose
isomerase from Thermus aquaticus HB8 Biol Chem Hoppe Seyler
371, 527 – 536.
27 Marg, G.A & Clark, D.S (1990) Activation of the glucose
isomerase by divalent cations: evidence for two distinct
metal-binding sites Enzyme Microb Technol 12, 367 – 373.
28 Jenkins, J., Janin, J., Rey, F., Chiadmi, M., van Tilbeurgh, H.,
Lasters, I., De Maeyer, M., Van Belle, D., Wodak, S.J., Lauwereys,
M., Stanssens, P., Mrabet, N.T., Snauwaert, J., Matthyssens, G &
Lambeir, A.-M (1992) Protein engineering of xylose (glucose)
isomerase from Actinoplanes missouriensis 1 Crystallography and
site-directed mutagenesis of metal binding sites Biochemistry 31,
5449 – 5458.
29 Allen, K.N., Lavie, A., Glasfeld, A., Tanada, T.N., Gerrity, D.P.,
Carlson, S.C., Farber, G.K., Petsko, G.A & Ringe, D (1994) Role
of the divalent metal ion in sugar binding, ring opening, and
isomerization by D -xylose isomerase: replacement of a catalytic
metal by an amino acid Biochemistry 33, 1488 – 1494.
30 Hartley, B.S., Hanlon, N., Jackson, R.J & Rangarajan, M (2000)
Glucose isomerase: insights into protein engineering for increased
thermostability Biochim Biophys Acta 1543, 294 – 335.
31 Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman,
J.G., Smith, J.A & Struhl, K., eds (1993) Current Protocols in
Molecular Biology Greene Publishing & Wiley-Interscience, New York.
32 Boyer, H.W & Roulland-Dussoix, D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli J Mol Biol 41, 459 – 472.
33 Dische, Z & Borenfreund, E (1951) A new spectrophotometric method for the detection and determination of keto sugars and trioses J Biol Chem 192, 583 – 587.
34 Lambeir, A.M., Lauwereys, M., Stanssens, P., Mrabet, N.T., Snauwaert, J., van Tilbeurgh, H., Matthyssens, G., Lasters, I., De Maeyer, M., Wodak, S.J., Jenkins, J., Chiadmi, M & Janin, J (1992) Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis 2 Site-directed mutagenesis of the xylose binding site Biochemistry 31, 5459 – 5466.
35 Cha, J., Cho, Y., Whitaker, R.D., Carrell, H.L., Glusker, J.P., Karplus, P.A & Batt, C.A (1994) Perturbing the metal site in
D -xylose isomerase Effect of mutations of His-220 on enzyme stability J Biol Chem 269, 2687 – 2694.
36 Dawson, R.M., Elliott, D.C., Elliott, W.H & Jones, K.M., eds (1986) Data for Biochemical Research, 3rd edn Oxford University Press, London.
37 Scheler, A., Rygus, T., Allmansberger, R & Hillen, W (1991) Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus licheniformis encoded regulon for xylose utilization Arch Microbiol 155, 526 – 534.
38 Hess, J.M., Tchernajenko, V., Vieille, C., Zeikus, J.G & Kelly, R.M (1998) Thermotoga neapolitana homotetrameric xylose isomerase is expressed as a catalytically active and thermostable dimer in Escherichia coli Appl Environ Microbiol 64,
2357 – 2360.
39 Callens, M., Tomme, P., Kersters-Hilderson, H., Cornelis, R., Vangrysperre, W & De Bruyne, C.K (1988) Metal ion binding to
D -xylose isomerase from Streptomyces violaceoruber Biochem J.
250, 285 – 290.
40 Callens, M., Kersters-Hilderson, H., Vangrysperre, W & De Bruyne, C.K (1988) D-xylose isomerase from Streptomyces violaceoniger: structural and catalytic roles of bivalent metal ions Enzyme Microb Technol 10, 695 – 700.
41 van Bastelaere, P., Vangrysperre, W & Kersters-Hilderson, H (1991) Kinetic studies of Mg(2þ)-, Co(2þ)- and Mn(2þ)-activated
D -xylose isomerases Biochem J 278, 285 – 292.
42 Collyer, C.A., Henrick, K & Blow, D.M (1990) Mechanism for aldose-ketose interconversion by D -xylose isomerase involving ring opening followed by a 1,2-hydride shift J Mol Biol 212,
211 – 235.
43 Batt, C.A., Jamieson, A.C & Vandeyar, M.A (1990) Identification
of essential histidine residues in the active site of Escherichia coli xylose (glucose) isomerase Proc Natl Acad Sci USA 87,
618 – 622.
44 Varsani, L., Cui, T., Rangarajan, M., Hartley, B.S., Goldberg, J., Collyer, C & Blow, D.M (1993) Arthrobacter D -xylose isomerase: protein-engineered subunit interfaces Biochem J 291, 575 – 583.
45 Sudfeldt, C., Schaffer, A., Kagi, J.H., Bogumil, R., Schulz, H.P., Wulff, S & Witzel, H (1990) Spectroscopic studies on the metal-ion-binding sites of Co(2þ)-substituted D -xylose isomerase from Streptomyces rubiginosus Eur J Biochem 193, 863 – 871.
46 Bogumil, R., Huttermann, J., Kappl, R., Stabler, R., Sudfeldt, C & Witzel, H (1991) Visible, EPR and electron nuclear double-resonance spectroscopic studies on the two metal-binding sites of oxovanadium (IV)-substituted D -xylose isomerase Eur J Biochem.
196, 305 – 312.
47 Haney, P.J., Stees, M & Konisky, J (1999) Analysis of thermal stabilizing interactions in mesophilic and thermophilic adenylate kinases from the genus Methanococcus J Biol Chem 274, 28453– 28458.
48 Chakravarty, S & Varadarajan, R (2000) Elucidation of