It retained a consid-erable amount of activity from low concentrations to at least 0.6 m NaCl, and was adapted to work at higher salt concentrations than VcEndA by maintaining a low Km v
Trang 1stability of endonuclease I from Vibrio salmonicida
and Vibrio cholerae
Laila Niiranen1, Bjørn Altermark2, Bjørn O Brandsdal2, Hanna-Kirsti S Leiros2, Ronny Helland2, Arne O Smala˚s2and Nils P Willassen1
1 Department of Molecular Biotechnology, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, Norway
2 Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, Faculty of Science, University of Tromsø, Norway
Extracellular and periplasmic enzymes of marine
organisms are exposed to environments in which
large variations in temperature and salinity can
occur Such conditions require the proteins to fold
effectively and maintain their stability in spite of the
stresses they face [1] At the same time, enzymatic
activity is dependent on fine-tuned structural
flexibi-lity [2] How do enzymes cope with these contradict-ing demands?
The study of the structural and functional adapta-tion of proteins from extremophilic organisms is an active research area, and several interesting observa-tions of the adaptive mechanisms have been made The extreme temperature stability of thermophilic
Keywords
endonuclease I; kinetics; salt adaptation;
thermodynamic stability; Vibrio
Correspondence
N P Willassen, Department of Molecular
Biotechnology, Institute of Medical Biology,
Faculty of Medicine, University of Tromsø,
N-9037 Tromsø, Norway
Fax: +47 776 453 50
Tel: +47 776 446 51
E-mail: nilspw@fagmed.uit.no
(Received 24 October 2007, revised 14
December 2007, accepted 1 February 2008)
doi:10.1111/j.1742-4658.2008.06317.x
Adaptation to extreme environments affects the stability and catalytic effi-ciency of enzymes, often endowing them with great industrial potential
We compared the environmental adaptation of the secreted endonuclease
I from the cold-adapted marine fish pathogen Vibrio salmonicida (VsEndA) and the human pathogen Vibrio cholerae (VcEndA) Kinetic analysis showed that VsEndA displayed unique halotolerance It retained a consid-erable amount of activity from low concentrations to at least 0.6 m NaCl, and was adapted to work at higher salt concentrations than VcEndA by maintaining a low Km value and increasing kcat In differential scanning calorimetry, salt stabilized both enzymes, but the effect on the calorimetric enthalpy and cooperativity of unfolding was larger for VsEndA, indicating salt dependence Mutation of DNA binding site residues (VsEndA, Q69N and K71N; VcEndA, N69Q and N71K) affected the kinetic parameters The VsEndA Q69N mutation also increased the Tm value, whereas other mutations affected mainly DHcal The determined crystal structure of VcEndA N69Q revealed the loss of one hydrogen bond present in native VcEndA, but also the formation of a new hydrogen bond involving residue
69 that could possibly explain the similar Tmvalues for native and N69Q-mutated VcEndA Structural analysis suggested that the stability, catalytic efficiency and salt tolerance of EndA were controlled by small changes in the hydrogen bonding networks and surface electrostatic potential Our results indicate that endonuclease I adaptation is closely coupled to the conditions of the habitats of natural Vibrio, with VsEndA displaying a remarkable salt tolerance unique amongst the endonucleases characterized
so far
Abbreviations
DSC, differential scanning calorimetry; EndA, endonuclease I; VcEndA, Vibrio cholerae endonuclease I; VsEndA, Vibrio salmonicida
endonuclease I; Vvn, Vibrio vulnificus endonuclease I.
Trang 2proteins is thought to be a result of extensive
intra-molecular networks and compact packing that restrict
their flexibility [3,4] Psychrophilic enzymes, in
contrast, are thermolabile and have been hypothesized
to use increased flexibility to cope with the increased
viscosity and decreased thermal vibrations at low
temperature [5,6] They may also use local flexibility
to maintain a functional active site, whilst separate
more rigid domains confer stability to their structure
[7] Compared with non-halophiles, halophilic proteins
have an excess of acidic amino acid residues that
create a negative surface potential and a protective
hydrated ion network [1,8] The charged surface is,
however, destabilizing, especially at low salt
concen-trations [9], and most halophilic proteins are
inacti-vated at NaCl or KCl concentrations below 2 m [1]
The structural basis of salt-tolerant activity remains
to be elucidated, although electrostatic interactions
have been implicated [10] The salinity of seawater is
significantly lower than that of extreme halophile
habitats, so that only milder forms of adaptation
may be necessary for periplasmic and extracellular
proteins of marine organisms
The functional characterization of extremophilic
proteins has so far focused on the obvious, i.e the
effects of temperature on thermophilic and
psychro-philic proteins, and salinity on halopsychro-philic proteins
However, many of the environments in which
extremo-philes thrive are extreme with respect to more than just
one parameter For example, in the field of
psychro-phile research, the majority of enzymes studied so far
have been extracellular and of marine origin [7], which
poses a problem when conclusions are to be drawn
about the mechanisms of cold adaptation Are the
observed adjustments a result of true adaptation to
low temperature, or a combination of cold and salt
adaptation? Choosing non-marine (freshwater)
psychro-philes as study targets has been proposed as a
solution [7] The interplay between the two types of
adaptation is, however, interesting in itself, and it is
possible to design experiments in a manner that
facili-tates the separation of the two effects The first steps
towards this approach have been taken In a
compara-tive study of a marine psychrophilic and an estuarine
mesophilic endonuclease I (EndA, EC 3.1.30) [11], the
different salt optima of the enzymes were taken into
consideration when the temperature-dependent
enzy-matic properties were characterized In the discussion,
the authors stressed the importance of performing
measurements in buffers that were as physiological as
possible Similar to psychrophilic EndA, marine
car-rageenase was found to display an activity optimum
around the salt concentration of seawater [12] It
appears that the choice of buffer and the determina-tion of the salt dependence of the activity are impor-tant in comparative experiments on extracellular enzymes
EndA is a periplasmic or extracellular sugar non-specific endonuclease Its physiological function is not known, but it has been proposed to be involved in the prevention of the uptake of foreign DNA, the degrada-tion of intestinal mucus to facilitate colonizadegrada-tion, and the provision of nucleotides for the cells [13] Although EndA has been isolated from many pathogenic bacte-ria, it does not appear to be involved in virulence [13– 15] It may, however, affect the bacterial survival rate through the degradation of neutrophil extracellular traps in mammals, and possibly also in fish [16,17] The structures of three Vibrio endonucleases, V sal-monicida (VsEndA) [18], V cholerae (VcEndA) [19] and V vulnificus (Vvn) [20], are available, and also the structure for Vvn bound to 8 bp and 16 bp dsDNA [20,21] A reaction mechanism has been proposed based on the protein–DNA complex structure [20] Sequence identity between mature Vvn and VcEndA is 75% and between Vvn and VsEndA is 74% The structural fold and the active site containing the cata-lytically important His80 are identical in all three structures Temperature adaptation has not been found
to affect the reaction mechanism of any homologous enzymes studied so far [22] The thermal adaptation of VsEndA and VcEndA has been studied previously, revealing that VsEndA has a higher kcat value at 5–37 C and is less thermostable than VcEndA [11] The shape-complementary surface of Vvn contacts the DNA only at the backbone phosphate groups [20] Comparisons of Vvn, VsEndA and VcEndA have revealed that most of the charged residues in the bind-ing cleft are conserved [18] The exceptions to this are two interesting regions with high-temperature B-factors pointed out by Altermark et al [18] The first is loop 51–54 which contains two more positive charges in VsEndA than in VcEndA, but is unlikely to contact DNA The second is residues 69 and 71 which partici-pate in the formation of the substrate binding site These residues are Gln and Lys, respectively, in VsEndA, but both are replaced by Asn in VcEndA Intuitively, such changes in charge and steric effects may alter substrate binding and salt sensitivity
In this study, the effect of NaCl concentration on the kinetic constants and thermodynamic stability of VsEndA and VcEndA was investigated In addition, the effects of reciprocal mutations of two non-con-served DNA binding site residues (VsEndA, Q69N and K71N; VcEndA, N69Q and N71K) on the kinetics, thermostability and salt dependence of these enzymes
Trang 3were examined Although salt stabilizes both native
enzymes equally, VsEndA is adapted to retain activity
at much higher salt concentrations than VcEndA The
relationship between these observations and the
struc-ture determined for the VcEndA N69Q variant, as well
as the previously published native EndA structures, is
discussed A thorough decomposition of the
thermo-dynamic data, together with mutational and structural
investigations, was used to gain an insight into
halotol-erant adaptation
Results
Protein production and thermal stability
The VsEndA variants Q69N and K71N and the
VcEndA variants N69Q and N71K were expressed in
a soluble form at levels comparable with the native
endonucleases The mutations did not change the
puri-fication properties of the enzymes
The effect of NaCl on the thermal stability of
VsEndA and VcEndA was investigated by
perform-ing differential scannperform-ing calorimetry (DSC) scans in
the presence of different salt concentrations The
thermal stabilities of the mutated enzymes were
determined at a single salt concentration (0.175 m
for VcEndA and 0.425 m for VsEndA variants)
cho-sen on the basis of the optimal activity conditions of
the native enzymes [11] The denaturation peaks were
symmetrical, except for some exothermic distortion
of the thermograms after the denaturation peak,
especially in the VcEndA sample with 0.050 m NaCl
Visible aggregation was present in most samples after
the thermal scan, and refolding experiments were not
pursued As shown in Fig 1, NaCl stabilized both
VsEndA and VcEndA by increasing the Tm value
Salt also affected the shape of the thermograms,
making the denaturation peaks narrower and sharper
with increasing salt concentration DSC of VsEndA
in 0.050 m NaCl was not performed because of
sam-ple instability
The symmetrical shape of the thermograms suggests
that the transition proceeds via a single transition
state The increases in Tm from 0.175 to 1 m NaCl
were 10.1 and 9.0C for VsEndA and VcEndA,
respectively (Table 1) The DHcalvalues increased with
salt concentration, except for VcEndA above 0.425 m
NaCl, although DHeff also increased in this case
VsEndA Q69N showed a higher Tm value, but DHcal
was unchanged All other mutants showed Tm values
comparable with the native enzyme, but a lower DHcal
The accordance between DHcal and the
model-depen-dent van’t Hoff enthalpy (DHeff) was best at moderate
salt concentrations, decreasing at high extreme concen-trations and for the mutants The denaturation heat capacity increment could not be determined because of irreversibility of unfolding
Kinetics Kinetic measurements were made in 0–0.6 m NaCl Striking differences were observed in the Km and kcat values of the native endonucleases (Fig 2) The Km value for VcEndA increased steeply at salt concentra-tions above 0.25 m An equivalent increase was seen for VsEndA above 0.50 m NaCl The kcat values also showed the same salinity optima: 0.25 m for VcEndA and 0.50 m for VsEndA VsEndA was increasingly more efficient than VcEndA in terms of kcat⁄ Km (Table 2) as the salt concentration increased The
kcat⁄ Kmsalt optima were not very different for the two
Fig 1 Denaturation heat capacity curves of the native and mutant VsEndA (top) and VcEndA (bottom) Differential scanning calorime-try profiles were recorded at a scan rate of 1 CÆmin)1in a buffer containing 0.175, 0.425 and 1.00 M NaCl for native VsEndA, and also 0.050 M NaCl for native VcEndA For VsEndA and VcEndA mutants, 0.425 and 0.175 M NaCl, respectively, were used Thermograms were baseline-subtracted and normalized for protein concentration.
Trang 4enzymes (0.175 and 0.1 m for VsEndA and VcEndA,
respectively), but the optimum was much broader for
VsEndA
The reciprocal mutations of the two residues partici-pating in creating the substrate binding site affected both kinetic constants (Table 2) The variants dis-played higher Km and kcat values, especially at high salt concentrations, except for the VcEndA N71K mutation which showed a decreased kcat value and a minimal effect on Km The catalytic efficiency of all variants was decreased compared with the native enzymes Interestingly, the salt optimum of VcEndA N69Q was shifted to zero salinity; both VsEndA vari-ants were also more efficient than the native enzyme at zero salinity
Structure of VcEndA N69Q The crystal structure of VcEndA N69Q was deter-mined to 1.7 A˚ resolution, and data collection and refinement statistics are presented in Table 3 The elec-tron density was well defined for most of the protein chain, and the mutated structure was similar to the native VcEndA with an rmsd of 0.20 A˚ for main chain atoms Differences were found for Asn71 and the mutated residue 69 Electron density maps (Fig 3) showed that the orientation of these side chains was different from the native structure The side chain of Gln69 in VcEndA N69Q was rotated away from resi-due 72 and was unable to form the Asn69 OD1– Arg72 N hydrogen bond, which has been suggested to stabilize the 69–72 loop in VcEndA [18] Instead, Gln69 NE2 formed a hydrogen bond with Asn129 OD1 and a water-mediated hydrogen bond with Glu125 O Gln69 OE1 in VcEndA N69Q interacted through water molecules to both the side chain of Arg67 and to Glu125 O The orientation of Arg67 was slightly shifted relative to the native structure Interest-ingly, the side chain of Asn71 in the mutated structure was also rotated, interacting only with a symmetry related molecule with a hydrogen bond to Glu179 O and a water-mediated bond to Gln180 O
Electrostatic calculations The electrostatic surface potentials of the enzyme vari-ants were calculated at the optimal salinity of the native enzymes (Fig 4) The effects of the mutations
on the overall potentials were small, but some local changes were observed The mutations of VsEndA appeared to result in a less positively charged surface
by increasing the exposure of a negatively charged patch (VsEndA Q69N, Fig 4B) or through the loss of
a positive charge (VsEndA K71N, Fig 4C) VcEndA N69Q mutation (Fig 4E) led to the rotation of a neighbouring positive charge, Arg67, whereas, in
Table 1 Thermodynamic parameters of the thermal unfolding of
VsEndA and VcEndA as a function of NaCl concentration
deter-mined by DSC.
NaCl
( M )
Tm (C)
DHcal (kJÆmol)1)
DHeff (kJÆmol)1) DHcal⁄ DH eff
VsEndA a
VcEndAa
a
Molecular masses: VsEndA, 25 005 Da; VcEndA, 24 732 Da;
VsEndA mutants, 24 645 Da; VcEndA mutants, 24 991 Da.b
Mini-mal values as a result of aggregation.
Fig 2 Plot of the kinetic parameters Km(A) and kcat(B) for native
VsEndA (d) and VcEndA (s) in 0–0.6 M NaCl The error bars
repre-sent maximum and minimum values.
Trang 5VcEndA N71K (Fig 4F), an increased positive surface
potential was observed
Discussion
For marine organisms and their extracellular proteins,
adaptation to environmental conditions can be
assumed to be somewhat more complex than simple
temperature or salt adaptation Previous studies of the
two secreted endonucleases VsEndA (marine
psychro-philic) and VcEndA (estuarine mesopsychro-philic) have shown
that their activity is strongly dependent on
tempera-ture, but also on NaCl concentration [18] We studied
how different salt concentrations and mutations affect
the stability and kinetic constants of VsEndA and
VcEndA, and found the effects to be striking,
espe-cially for VsEndA
Thermal stability
At 175 mm NaCl, the native enzymes display a
differ-ence of 11.1C in Tm The difference in Tm and the
calorimetric enthalpy is small compared with that
found for extremophilic DNA ligases [23] This
con-firms our previous finding that, for a psychrophilic
enzyme, VsEndA has a relatively high temperature
optimum and kinetic stability [11] The reason for the
small DTm may be linked to the charged residues, as the hydrophobic cores of the two enzymes are similar The extra salt bridges in the C-terminus of VcEndA and the smaller repulsion between the positively charged residues are the likely cause for the increased
Tmvalue compared with VsEndA
At concentrations less than 1 m, salt interacts with proteins in a non-specific manner by neutralizing charges The addition of salt may lead to a decrease in intramolecular electrostatic repulsion, but an increase
in the hydrophobic effect [24,25] Quantitative studies
of the effects of NaCl on protein thermostability are scarce, but, in general, it has been found that there is a direct relationship between salinity and the upshift in the thermal unfolding temperature Tm [26–28] This agrees with our finding of a nearly equal increase in the Tm values of the two enzymes when salt is added, although the salt-induced increase in enthalpy is more pronounced in VsEndA than in VcEndA
A salt-induced increase in Tm with a simultaneous decrease in DHcal has been proposed to result from stronger but less cooperative intramolecular interac-tions [29] In this context, cooperativity means that the protein structure unfolds as a single unit (one single transition), as opposed to several more or less inde-pendent units (several transitions) The increase in Tm and DHcal of EndA with increasing salt concentration
Table 2 Kinetic constants for native and mutant VsEndA and VcEndA at 0–0.6 M NaCl.
[NaCl] ( M ) VsEndA VsEndA Q69N VsEndA K71N VcEndA VcEndA N69Q VcEndA N71K VsEndA ⁄ VcEndA
0.100 32.0 ± 5.4 82.1 ± 10.7 82.5 ± 12.0 44.1 ± 4.5 283 ± 29 78.6 ± 8.8 0.72
0.500 356 ± 16 2540 ± 400 1760 ± 280
0.600 1150 ± 100
k cat (s)1) 0 2.77 ± 0.11 4.82 ± 0.16 3.83 ± 0.12 2.40 ± 0.07 2.98 ± 0.08 1.62 ± 0.04 1.2
0.100 3.88 ± 0.15 7.64 ± 0.29 7.93 ± 0.34 4.05 ± 0.09 5.82 ± 0.22 2.88 ± 0.08 0.96 0.175 7.35 ± 0.25 12.4 ± 0.5 9.73 ± 0.33 5.75 ± 0.21 6.94 ± 0.15 4.18 ± 0.13 1.3
0.250 9.90 ± 0.25 23.8 ± 1.2 12.1 ± 0.5 6.39 ± 0.13 8.98 ± 0.88 4.68 ± 0.29 1.5
0.350 15.8 ± 0.5 29.4 ± 1.2 25.0 ± 0.6 4.43 ± 0.33 7.25 ± 1.33 4.86 ± 0.48 3.6
0.500 22.4 ± 0.4 53.3 ± 5.8 41.5 ± 4.2
0.600 18.6 ± 0.9
Trang 6could therefore be interpreted, conversely, as increased
cooperativity of unfolding and a more compact
struc-ture as a result of stronger intramolecular interactions
These salt-induced effects on DHcal and DHeff are
stronger in VsEndA, possibly because of the increased
number of solvent-exposed charged and hydrophobic
residues relative to VcEndA, as found when viewing
the molecular surfaces and their amino acid properties
This indicates a certain degree of salt dependence of
VsEndA stability, but is in disagreement with the
observation that the Tm values for both enzymes are
equally affected by salt addition It has been suggested
that salt stabilizes halophilic proteins to a greater
extent than non-halophilic proteins, and that halophilic
proteins are destabilized by low salt concentrations [9]
Both effects may originate from the characteristic high
negative surface potential of halophilic proteins and
increased solvent ion binding [8,30,31] The equal
increase in Tm of VsEndA and VcEndA may suggest that VsEndA does not have any specific ion binding sites on its surface relative to VcEndA, and is not halo-philic The more highly charged surface of VsEndA may, however, constitute a more cooperative solvent ion binding network, which makes it possible for the enzyme to better tolerate fluctuations in salt concentra-tion
The observed general decrease in DHcal⁄ DHeff with increasing salt concentration may imply that the theo-retical model used is unable to tackle the increased cooperativity of unfolding, but may also be explained
by an increase in the degree of irreversible unfolding (Table 1) The reversibility of thermal unfolding of a halophilic b-lactamase has been found to be inversely dependent on salt concentration, and has been pro-posed to be caused by the salting-out effect of NaCl [28] NaCl can neutralize the surface charges of unfolded proteins and facilitate aggregation
Kinetics The release of coordinated ions and water molecules from the solvation shells of enzymes and substrates provides a positive entropic effect that drives substrate binding This effect is dependent on both temperature and salt concentration [32,33] At elevated salt concen-trations or low temperatures, the gain in entropy on release of ions is reduced and substrate binding is therefore weaker [33] This makes binding of highly charged DNA very challenging for marine enzymes DNA binding to non-halophilic proteins has been found to be inversely dependent on salt concentration [32,34], whereas the binding efficiency of halophilic proteins appears to actually increase with increasing salt concentration [35,36] A halophilic nuclease from Micrococcus varians [37] with maximal activity in 3–4 m NaCl displays an excess of acidic residues char-acteristic of many halophilic enzymes It is possible that this enzyme has a binding mechanism involving counterion uptake, similar to that proposed for the halophilic Pyrococcus woesei TATA-box binding pro-tein [36] Contrary to these halophilic proteins, VsEndA displays an excess of basic residues contacting the negatively charged substrate, and the Km value increases with increasing salt concentration, although this occurs at a much higher salinity than for VcEndA
In our previous study of EndA temperature adapta-tion, the more positively charged surface of VsEndA was considered not to decrease the Km value relative
to VcEndA [11] These measurements were made at the respective optimal salt concentrations of the enzymes, where the Kmvalues were found to be of the
Table 3 X-ray data collection and crystallographic refinement
sta-tistics for the VcEndA N69Q structure.
Data collection
c = 75.64 Resolution (A ˚ ) (highest bin) 25.00-1.70 (1.79-1.70)
Mean (<I> ⁄ <rI>) 12.8 (2.2)
Refinement
R-factor (all reflections) (%) 19.7
No of other molecules 1 Mg 2+ , 1 Cl)
Average B-factor (A˚2 )
Water molecules ⁄ Mg 2+ ⁄ Cl) 24.4 ⁄ 22.7 ⁄ 11.1
Ramachandran plot
Most favoured regions (%) 93.9
Additionally allowed regions (%) 5.5
Generously allowed regions (%) 0.6
a R-sym = ( P
h
P
I | Ii(h) – <I(h)> |) ⁄ ( P
h
P
I I(h)), where Ii(h) is the ith measurement of reflection h and <I(h)> is the weighted mean of all
measurements of h. b5% of the reflections were used in the
R-free calculations.
Trang 7same magnitude This can be explained by the similar
or slightly lower electrostatic surface potential of
VsEndA compared with VcEndA at the respective
optimal salinities (Fig 4A,D) The results of the
pres-ent study show that the Kmvalues are strongly affected
by the NaCl concentration, similar to the surface
charge of the enzyme The higher positive charge of
VsEndA therefore decreases Km, but this is a method
of coping with the charge shielding of buffer solutes
rather than low temperatures The higher charge may
allow VsEndA to retain sufficient charge, even at
rela-tively high salinity, to enable tight substrate binding,
contrary to VcEndA
The salt adaptation of kinetic constants as striking
as that observed in the present study has not been
pre-sented previously Only two comparative studies of the
dependent kinetics of a non-halophilic and a
salt-adapted enzyme have been published to date In the
comparison of halotolerant Dunaliella salina carbonic
anhydrases dCA I and dCA II and the human
homo-logue in 0–0.5 m NaCl, the largest differences were
found in the Km values [10,38] Similar to our results,
the halotolerant enzymes retained a low Km, whereas
the Km value of the non-halophilic enzyme increased
considerably with the addition of salt These results imply that Km salt tolerance is a feature typical to halotolerance The role of kcat is less clear Both Bageshwar et al [38] and Premkumar et al [10] found
kcat to be increased only slightly by salt, whereas we observed a large effect for kcat for both VsEndA and VcEndA at high salinity The higher catalytic rate may reflect the dependence of kcaton the substrate binding and dissociation rate constants, as found in the cold adaptation of cod trypsin [39], or, in the case of VsEndA, may be linked in some way to cold adapta-tion, where an increase in kcat is a typical mechanism [7] A high kcatvalue may also be a feature of salt tol-erance, but more studies on halotolerant enzymes are required to verify this The addition of salt may cause the EndA substrate binding cleft to reach a more opti-mal configuration for enzyme catalysis, thereby affect-ing kcat The DSC thermograms indicate that salt constricts the structural fluctuations of the enzyme At
a certain concentration, these fluctuations may become optimal for enzymatic turnover, whereas, at salt con-centrations above the optimum, the structure becomes too rigid and will function less optimally If the stabi-lizing effect of salt is caused mainly by the weakening
Fig 3 (A) Electron density (2Fo– Fcat 1r contoured in blue) and omit (Fo– Fcat 3r contoured in green) maps illustrating the orientation of Asn71 and the N69Q mutation in the VcEndA N69Q structure (B) Superposition of the VcEndA N69Q mutant (red), native VcEndA (blue) and VsEndA (green) structures (C) A partial sequence alignment of VsEndA and VcEndA The asterisks indicate the non-conserved residues selected for mutagenesis, and the plus sign denotes the catalytically important His80 Sequence numbering follows that of Vvn [20].
Trang 8of repulsive charges, it is reasonable to imagine that
VsEndA must be screened by a higher salt
concentra-tion than VcEndA to be able to funcconcentra-tion optimally
Effects of mutations
The point mutations are not in the immediate vicinity
of the active site situated at the bottom of the
posi-tively charged pocket, but are still likely to affect the
shape, stability and charge of the DNA binding site
(Fig 4) The Asn69 side chain in VcEndA forms a
hydrogen bond to Arg72 N, which may stabilize this loop region relative to Vvn and VsEndA [19], whereas the hydrogen bond observed in the VcEndA N69Q structure (Gln69 to Asn129) stabilizes other regions The characterization of the VcEndA N69Q mutant (Table 2) shows higher Km and kcat values compared with native VcEndA The lost hydrogen bond (from 69
to 72) in VcEndA N69Q may increase the flexibility of the 69–72 loop, possibly explaining the decreased bind-ing affinity and increased catalytic rate In addition, the shape of the DNA binding pocket in VcEndA
A
B
C
D
E
F
Fig 4 Electrostatic surface potentials in the DNA binding groove of VsEndA with a modelled DNA (A), VsEndA Q69N (B) and VsEndA K71N (C) all in 0.425 M NaCl, and VcEndA (D), VcEndA N69Q (E) and VcEndA N71K (F) all in 0.175 M NaCl The black arrows show the mutated residues The sur-face potential is coloured from )10 kT ⁄ q (red) to 10 kT ⁄ q (blue).
Trang 9N69Q is slightly altered as both residues 71 and 69 are
moved in the crystal structure (Fig 3), and the current
orientation of Gln69 is different from the Vvn–DNA
structure (Fig 3B) and very close to a modelled DNA
backbone, possibly explaining the higher Km values
(Table 2) Gln69 in VsEndA is poorly defined in the
native crystal structure, and the characterization of the
VsEndA Q69N mutant (Table 2) reveals poorer DNA
binding and an increased kcat with a maximum at
0.5 m NaCl The Gln69 side chain in the Vvn–DNA
structure (PDB 1OUP) is less than 3.2 A˚ from the
DNA backbone, and mutation to the shorter Asn in
VsEndA Q69N may prevent the formation of
favour-able DNA–enzyme interactions, and lead to the higher
Km values observed In addition, the Asn69 to Arg72
hydrogen bond lost in the VcEndA N69Q structure
may be formed in VsEndA Q69N, although this
should be verified by structural studies This additional
hydrogen bond may explain the increased stability of
the VsEndA Q69N mutant, and the subsequent
decrease in flexibility may further impair substrate
binding and contribute to the higher Kmvalues
The introduction or removal of a positively charged
residue (N71K and K71N) has a large effect on the
electrostatic surface potential (Fig 4) However, the
VcEndA N71K mutant has a binding affinity and
turnover comparable with the native VcEndA Being
more distal from DNA, as observed in the Vvn–DNA
structure, residue 71 may have less influence on DNA
binding than residue 69 Interestingly, kcat starts to
decrease when the salt concentration exceeds 0.25 m
for both native VcEndA and VcEndA N69Q, but this
is not observed for the VcEndA N71K variant The
VsEndA K71N mutant shows poorer DNA binding
and increased kcat compared with VsEndA, indicating
that the positive charge is more important for DNA
binding in VsEndA than in VcEndA
No side chain contacts are seen for residue 71 in
the native structures or models of the mutants As the
longer side chain of lysine has more rotamers, the
N71K substitution in VcEndA may stabilize the
struc-ture by increasing the rotational entropy, whilst
retain-ing the backbone interactions An increase of 1C is
observed for the Tm value of this mutant In VsEndA
K71N, both DHcal and the cooperativity of unfolding
are decreased, possibly indicating changes in the
hydrogen bonding networks The increase in Km may
be the result of a slightly enlarged binding site or less
positive charge Indeed, the changes seen in the
electro-static surface potential of each of the mutants (Fig 4)
match surprisingly well with their kinetic results Both
VsEndA mutants and the VcEndA N69Q mutant show
more dispersed or less positive charge, and,
accord-ingly, display higher and more salt-sensitive Kmvalues VcEndA N71K does not display a lower Kmvalue, but one similar to the native enzyme, in spite of the acqui-sition of an additional positive charge, possibly because of other effects caused by the mutation Even minor changes in protein structure, such as single amino acid replacements, can induce a signifi-cant change in the cooperativity of unfolding, and be detected as changes in the effective (van’t Hoff) enthalpy [40] In the DSC experiments, only the VsEndA Q69N mutation had the expected effect, increasing the stability via both Tmand the cooperativ-ity of unfolding, although the DHcalvalue was compa-rable with that of the native enzyme Whether or not local changes are reflected in the global hydrogen bonding networks, and how widespread are their effects, cannot be discerned from the native and mutant crystal structures Effects on hydrogen bonding networks may change the electron density distribution around and in the active site and, together with small conformational alterations, may speed up the rate-lim-iting step of hydrolysis Such long-range effects have been proposed to be the cause of more mesophilic-like kinetic behaviour in psychrophilic a-amylase, where single amino acid mutations were introduced outside the catalytic cleft [22,41] The identity of the rate-limit-ing step in the endonuclease reaction mechanism is not known However, the high catalytic efficiency of both VsEndA and VcEndA (kcat⁄ Km in the region of
108s)1Æm)1) shows that the reaction is nearly diffusion controlled, suggesting that the rate-limiting step is either substrate binding or dissociation As all muta-tions affect Km, especially at high salt concentrations, the optimization and salt tolerance of binding interac-tions are most probably hampered by the mutainterac-tions
by electrostatic, steric or flexibility effects The less tight binding of DNA may enable the enzymes to release the products more easily, thus leading to the observed increase in the kcatvalues of three of the vari-ants Similarly, the seven-fold higher kcat value of the hyperactive variant of Escherichia coli dihydrofolate reductase, compared with the wild-type enzyme, has been suggested to result from increased flexibility and size of the substrate binding cleft, leading to an increased product dissociation constant [42]
Conclusions
The experiments conducted in this study show that the secreted endonuclease VsEndA from the marine psy-chrophilic V salmonicida is remarkably salt tolerant and therefore unique amongst the endonucleases char-acterized so far Salt has striking effects on the kinetic
Trang 10constants of VsEndA, and the high positive charge of
VsEndA is considered to be essential in counteracting
the charge shielding of buffer solutes and maintaining
a low Km at high salinity It is possible that Km salt
tolerance will emerge as a general feature for
halotoler-ant proteins The role of the high kcatvalue observed
for VsEndA is less clear, and more studies on
halotol-erant enzymes are required to elucidate this further
The salt-induced increase in enthalpy and cooperativity
of unfolding is more pronounced in VsEndA This
effect indicates the formation of a more compact
struc-ture through the strengthening of intramolecular
inter-actions or the weakening of intramolecular repulsive
forces, and the salt dependence of VsEndA stability
The higher positive electrostatic surface potential of
VsEndA compared with VcEndA plays a key role in
adaptation On the whole, the characteristics of
VsEndA and VcEndA illustrate the fine-tuned
adapta-tion to their natural environments
Materials and methods
Site-directed mutagenesis and plasmid
purification
Residue targets for mutagenesis were selected on the basis
of the sequence and structural alignments of Vvn, VsEndA
and VcEndA The selected residues 69 and 71 were
non-conserved between VsEndA and VcEndA, located in the
DNA binding region and close to the active site
direc-ted mutagenesis was performed using a QuikChange
Site-Directed Mutagenesis Kit (Stratagene, Cedar Creek, TX,
USA), as described in the manual The oligonucleotides
were synthesized by Sigma-Aldrich (St Louis, MO, USA)
Mutated plasmids were transformed into E coli TOP10
cells (Invitrogen, Carlsbad, CA, USA), and plasmid
extrac-tion was performed using QIAprep minipreps (Qiagen,
Hilden, Germany) or the alkaline lysis method [43]
Expression and purification
The expression and purification of recombinant VsEndA
and VcEndA native enzymes and mutants were performed
as described previously [11] with a few modifications Cells
were cultured in either shake-culture flasks or a Techfors S
fermenter (Infors, Bottmingen, Switzerland) The culture
temperature was kept at 37C until glucose was depleted,
after which the temperature was adjusted to 22C before
expression was induced The cells were harvested when they
reached the stationary phase and were collected by
centrifu-gation For periplasmic fractionation, the cells were
resus-pended in a 1 : 10 culture volume of fractionation buffer,
and incubated on ice for 1–1.5 h before the supernatant
was collected
Enzyme assay Enzyme activity measurements were assayed in triplicate at
23C in 75 mm Tris ⁄ HCl, pH 8.0 and pH 8.5 (VcEndA and VsEndA, respectively), 5 mm MgCl2 and 0–0.6 m NaCl Eight different concentrations (12–1470 nm) of DNaseAlert substrate (DNaseAlert QC System Kit; Ambion, Austin,
TX, USA) were used for the kinetic measurements, and
200 nm substrate for the other activity measurements The total reaction volume was 100 lL and reactions were started
by the addition of 10 lL of enzyme diluted in reaction buffer Protein LoBind tubes from Eppendorf (Hamburg, Germany) were used for enzyme dilutions because of the sticky nature of the enzyme The detailed assay procedure
is described elsewhere [11] sigmaplot software (Systat Software, San Jose, CA, USA) was used for data analysis, and Vmax and Km values were calculated by fitting the velocity data to the Michaelis–Menten equation
Differential scanning calorimetry Differential scanning calorimetry experiments were con-ducted on a Nano-Differential Scanning Calorimeter III, model CSC6300 (Calorimetry Sciences Corporation, Lin-don, UT, USA) Preparations of the native enzymes were first filtered with a 0.45 lm Spin-X centrifuge tube filter (Corning, Corning, NY, USA), and then dialysed overnight
at 4C against 1 L of dialysis buffer (50 mm Hepes, 5 mm MgCl2, pH 8.0) containing 0.050, 0.175, 0.425 or 1.00 m NaCl Slide-A-Lyzer dialysis discs from Pierce (Rockford,
IL, USA) with a 2 kDa cut-off were used The protein con-centration of the dialysed enzyme solution was determined using BioRad Protein Assay Dye Reagent Concentrate (BioRad, Hercules, CA, USA) with bovine serum albumin (Sigma) as standard The dialysates were used as blank ref-erences in DSC runs Reference buffers and samples were carefully degassed before loading into the DSC cells The scans were performed at a constant pressure of 304 kPa in the range 15–75C or 20–80 C with a heating rate of
1CÆmin)1 Thermograms were analysed according to a single non-two-state transition model in which Tm, DHcal and DHeff were fitted independently using cpcalc software (Calorimetry Sciences Corporation)
Crystallization, data collection and structure determination
The mutant VcEndA N69Q was crystallized in similar con-ditions as native VcEndA [19] using the hanging drop vapour-diffusion technique at room temperature with 6.2 mgÆmL)1 of protein in 50 mm Tris⁄ HCl pH 8.0, 5 mm MgCl2and 0.6 m NaCl Drops were made by mixing 1 lL
of protein with 1 lL of reservoir solution consisting of 0.1 m sodium acetate, 0.3 m ammonium acetate, 10 mm magnesium sulphate and 26% PEG8000 Crystals of about