Exploring the role of a glycine cluster in cold adaptationof an alkaline phosphatase Konstantinos Mavromatis1,*, Iason Tsigos2,*, Maria Tzanodaskalaki2, Michael Kokkinidis1,3 and Vassili
Trang 1Exploring the role of a glycine cluster in cold adaptation
of an alkaline phosphatase
Konstantinos Mavromatis1,*, Iason Tsigos2,*, Maria Tzanodaskalaki2, Michael Kokkinidis1,3
and Vassilis Bouriotis1,2
1
Department of Biology, Division of Applied Biology and Biotechnology, University of Crete, Greece;2Institute of Molecular Biology and Biotechnology (IMBB), Enzyme Technology Division, and the3Institute of Molecular Biology and Biotechnology,
Crystallography Division, Heraklion, Crete, Greece
In an effort to explore the role of glycine clusters on the cold
adaptation of enzymes, we designed point mutations aiming
to alter the distribution of glycine residues close to the active
site of the psychrophilic alkaline phosphatase from the
Antarctic strain TAB5 The mutagenesis targets were
residues Gly261 and Gly262 The replacement of Gly262 by
Ala resulted in an inactive enzyme Substitution of Gly261
by Ala resulted to an enzyme with lower stability and
increased energy of activation The double mutant G261A/
Y269A designed on the basis of side-chain packing criteria
from a modelled structure of the enzyme resulted in restor-ation of the energy of activrestor-ation to the levels of the native enzyme and in an increased stability compared to the mutant G261A It seems therefore, that the Gly cluster in combi-nation with its structural environment plays a significant role
in the cold adaptation of the enzyme
Keywords: alkaline phosphatase; psychrophiles; cold adaptation; structural flexibility; glycine clusters
Cold adapted enzymes, produced by organisms living in
permanently cold environments, exhibit a higher specific
activity at low temperatures [1–3] Moreover, this high
catalytic efficiency is consistently accompanied by a lower
thermal stability, although these properties are not always
correlated as shown by recent data from directed evolution
experiments which support the interdependence of these
properties [4–8]
The adaptation to cold is achieved through a decrease in
the activation energy, which results from an increased
protein flexibility, either of the whole protein or of a specific
domain in some multidomain proteins Furthermore,
evidence from the notothenioid A4-lactate dehydrogenases
support a cold adaptation model in which structural
flexibility increases are confined to small areas of the
molecule, thereby affecting the mobility of adjacent active
site structures and resulting in reduced energy barriers [9]
Therefore, psychrophilic adaptation seems to be associated
with localized rather than global increases in conformational
flexibility [10] This is in agreement with structural data,
which reveal that only minor modifications are necessary to
convert a mesophilic or thermophilic enzyme into a cold
adapted one [11–14]
Although the strategy of adaptation is unique to each
enzyme [15], it has been observed that amino-acid residues
involved in the catalytic mechanism are generally conserved
in psychrophilic and mesophilic enzymes [1] This suggests that generally the molecular basis of cold adaptation is associated with sequence changes outside the active site However, recent work from our group indicated that the psychrophilic character of an enzyme could also be altered
or masked by mutating active site residues [16] Several sequence patterns have been associated with psychrophilic adaptations, such as decreased levels of Pro and Arg residues, weakening of intramolecular interactions, increased solvent interactions, decreased charged residues interactions, and disulfide bonds [1,2,17] Increased levels of Gly residues or the establishment of Gly clusters have been frequently suggested to be associated with psychrophilicity [2] This could be a result of increased local structural flexibility due to the intrinsic flexibility of Gly residues [18] However, recent studies of Gly clusters [19] appear to contradict this assumption It seems that the correlation between the occurrence of Gly residues and the stability of proteins is complex as several parameters from the whole protein structure are involved and not just the intrinsic flexibility of Gly residues [20]
We have recently reported the cloning, sequencing and overexpression of the gene encoding alkaline phosphatase from the Antarctic strain TAB5 [16] Based on the crystal structure (at 2.4 A˚) of an Escherichia coli alkaline phospha-tase variant with a 28% amino-acid sequence identity to the psychrophilic enzyme, a three-dimensional model of the psychrophilic enzyme was constructed [21] We have also presented mutagenesis data that substantiate the role of the local flexibility on the psychrophilic character, and catalytic properties of the enzyme [16] In the case of alkaline phos-phatases, positions 261, 262 (in TAB5 alkaline phosphatase numbering) are often occupied by one Gly; this site is next
to one of the catalytic residues (Trp260 in the case of TAB5 alkaline phosphatase) In E coli and some Bacillus sp., there
Correspondence to V Bouriotis, Department of Biology,
Division of applied Biology and Biotechnology, University of Crete,
PO Box 1470, Heraklion 711 10, Crete, Greece.
Fax/Tel.: + 30 810 394375, E-mail: bouriotis@imbb.forth.gr
Abbreviation: pNPP, p-nitrophenyl phosphate.
Enzyme: alkaline phosphatase (EC 3.1.3.1).
*Note: these authors have equally contributed to this work.
(Received 12 December 2001, revised 14 March 2002,
accepted 18 March 2002)
Trang 2are no Gly residues at these positions In TAB5 alkaline
phosphatase, these two positions are both occupied by Gly
The presence of this Gly cluster in TAB5 alkaline
phosphatase has provoked us to explore its potential role
in the establishment of the psychrophilic properties of the
enzyme
E X P E R I M E N T A L P R O C E D U R E S
Materials
Restriction and DNA modification enzymes were
pur-chased from New England Biolabs (Beverly, MA, USA)
and MINOTECH (Heraklion, Greece) All chemicals
were of analytical grade for biochemical use PCR primers
were purchased from the Microchemistry Laboratory of
IMBB
Enzymatic assay
Alkaline phosphatase activity was followed
spectrophoto-metrically utilizing p-nitrophenyl phosphate (pNPP) as
substrate The release of product, p-nitrophenolate, was
monitored by measuring the absorbance at 405 nm using a
PerkinElmer photometer Specific activity was determined
in a buffer containing 1M diethanolamine/HCl (pH 10),
10% glycerol, 10 mM MgCl2, 1 mM ZnCl2, and 10 mM
pNPP at 20C Enzyme units were calculated as previously
described [22]
Steady-state enzyme kinetics
Steady-state enzyme kinetics were performed in the
tem-perature range 5–25C The program HYPER v 1.01 was
used for the determination of Vmaxand Kmvalues The kcat
values were calculated from Vmaxusing a molecular mass of
76 122 Da for the enzyme Reported values are the average
of three measurements The standard deviations do not
exceed 10% Thermodynamic parameters of the enzyme
were calculated as described previously [27]
Thermal inactivation of enzymes
In order to measure the thermal inactivation of enzymes,
they were incubated at 50C, in a buffer containing 1M
diethanolamine pH 10.0, 10 mM MgCl2, 1 mMZnCl2and
10% glycerol for different time periods and they were
subsequently incubated on ice for 30 min The remaining
activity was measured at 20C Reported values are the
average of at least two measurements The standard
deviations do not exceed 10%
Site-directed mutagenesis
Site directed mutagenesis was performed using standard
PCR methods [23] For the construction of the mutations
the following primers were synthesized: Gly261 to Ala,
upper primer 5¢-d(CAAATAGATTGGGCTGGCCATG
CAAATAAT)-3¢, lower primer 5¢-d(TATTTGCATGGCC
AGCCCAATCTATTTGAG)-3¢; Gly262 to Ala, upper
primer 5¢-d(ATAGATTGGGGTGCCCATGCAAATAA
TGCA)-3¢, lower primer 5¢-d(ATTATTTGCATGGGCA
CCCCAATCTATTTG)-3¢; Tyr269 to Ala, upper primer
5¢-d(TAATGCATCCGCTTTAATTTCTGAAATTA ATG)-3¢, lower primer 5¢-d(TCAGAAATTAAAGCGG ATGCATTATTTGCATG)-3¢
The upstream primer containing the NdeI restriction site (underlined) was: 5¢-d(GCTAGCATATGAAGCTTAAA AAAATTG)-3¢ and the downstream primer containing the EcoRI restriction site (underlined) was: 5¢-d(TTGAATTC GTTTATTGATTCCACTTTG)-3¢
The PCR reaction mixtures were incubated on an Eppendorf thermal cycler for 30 cycles of 94C for 1 min,
49C for 1 min, and 72 C for 1 min The amplified product was isolated by agarose gel electrophoresis, gel purified using QIAEX (Qiagen) and digested with NdeI and EcoRI restriction enzymes The resulting NdeI–EcoRI fragment was inserted into the pRSETA vector previously digested with these enzymes The ligation mixture was used
to transform competent cells of E coli strain XL1-MRF
Molecular modelling
A three-dimensional molecular model of the psychrophilic alkaline phosphatase was built [21] on the basis of the homology to the E coli enzyme the structure of which is known [24] For display of the model and for design and analysis of mutations the program SWISSPDB VIEWER was used [25]
Expression and purification of enzymes The protocol used for the expression of enzymes used has been previously described [16]
R E S U L T S
Choice of amino-acid substitutions Based on sequence comparisons, in most alkaline phospha-tases, the dipeptide corresponding to positions 261 and 262 (TAB5 numbering) contains one Gly residue; the second residue is usually Ala or His (Fig 1) In the E coli phosphatase these two positions are occupied by Gln and Asp, respectively Both positions are occupied by Gly residues in the TAB5 alkaline phosphatase This clustering
of Gly provides an interesting mutation target due to its potential relation to the psychrophilic character of the enzyme
Two point mutants were constructed; G261A and G262A where Gly261 and Gly262 were replaced by Ala,
Fig 1 Partial alignment of alkaline phosphatases at the region studied Mutation targets at positions 261, 262 and 269 of TAB5 alkaline phosphatase are shown in bold Grey boxes indicate corresponding residues in the other alkaline phosphatases.
Trang 3respectively By introducing an Ala residue in the place of
Gly it is expected that the conformational flexibility of the
main chain can be constrained with a minimum
perturba-tion of the local structure, resulting to a more rigid protein
(Fig 2) Moreover, Ala residues are common among
phosphatases at these positions (Fig 1)
On the basis of the molecular model [21] Ala261 is
expected to introduce steric clashes with the side chain of
Tyr269 (Fig 2B), which are not present in the structure of
the psychrophilic enzyme with the smaller Gly residue at
position 261 (Fig 2A) Replacement of Tyr269 by Ala in the double mutant G261A/Y269A is expected to remove most of the spatial constraints of the side chain interactions (Fig 2C)
Temperature dependence of activity in wild-type and mutant enzymes
The specific activity of all mutants was measured over the entire range of temperature (5–25C) where wild-type alkaline phosphatase is stable (Fig 3A) Mutant G262A had no significant activity at all temperatures tested making
it impossible to measure the specific activity or any kinetic parameters of this mutant We could only measure traces of activity after prolonged incubation (24 h)
The mutant G261A is more active at elevated tempera-tures (20–25C) compared to wild-type protein, while the mutant G261A/Y269A is less active at any given tempera-ture However, compared to the mesophilic enzyme from
E coli, these enzymes are approximately 10 times more active
Determination ofEaand thermodynamic parameters for wild-type and mutant enzymes
In order to elucidate the effect of mutations in terms of psychrophilic adaptation, we determined the energy of activation Eafor wild-type and mutant enzymes Figure 3B shows the Arrhenius plots for the temperature range of 10–25C The Ea of the enzymes reveal that the mutant G261A exhibits a higher value almost 2.5-fold higher than the native cold adapted enzyme (Table 1) The mutant G261A/Y269A exhibits an Eaalmost the same as in the case
of the native enzyme (T able 1)
Thermal inactivation of mutant and wild-type enzymes
In order to investigate the effects of mutations on the stability of psychrophilic alkaline phosphatase, the enzymes were incubated at 50C for different time periods and subsequently their remaining activity was measured As shown in Fig 3C, replacement of Gly261 by Ala in mutant G261A resulted in an enzyme with slightly lower stability
On the other hand, in the double mutant G261A/Y269A the additional replacement of Tyr269 by Ala restores the stability of the protein producing a more stable enzyme than the native one
D I S C U S S I O N
Recent studies have established that, adjustment of con-formational flexibility is essential for the temperature adaptation of enzymes [26] Moreover, localized increases
in conformational flexibility constitute an essential element
in cold adaptation [9] However, our incomplete under-standing of the relation between enzyme properties and conformational flexibility limits the exploitation of the full potential of protein engineering in the redesign of psychro-philic enzyme properties [15] In particular, the effects of local flexibility in psychrophilic enzyme properties have been so far studied only for regions, which indirectly affect the mobility of active site structures, but not for the active sites themselves [9]
Fig 2 Drawing of the three dimensional model of the wild type (A) and
mutant alkaline phosphatases G261A (B) and G261A/Y269A (C); only
residues that where studied are shown.
Trang 4In a previous study [16], we explored the possibility of
modifying the psychrophilic properties of an enzyme by
introducing, via mutagenesis, predictable flexibility changes
to key active site residues of the psychrophilic alkaline
phosphatase from the Antarctic strain TAB5 This
approach was based on an approximate homology-based
three-dimensional model of the psychrophilic enzyme and
sequence comparisons with mesophilic sequences The
mutagenesis targets were residues Trp260 and Ala219 of
the catalytic site and His135 of the Mg2+binding site The
most striking result was the loss of the psychrophilic
character of mutant W260K/A219N (as reflected by a
three-fold increase of the Ea value compared to the wild-type
enzyme) Interestingly, the activity of the mutant at elevated
temperatures (20–25C) exceeded that of the wild-type
protein Further substitution of His135 by Asp in the triple
mutant W260K/A219N/H135D restored a low energy of
activation In addition, the His135fi Asp replacement
resulted in a considerable stabilization of enzymes harboring
this mutation (single mutant H135D and triple mutant
W260K/A219N/H135D) These results suggested that the
psychrophilic character of mutants can be established or
masked by very slight variations of the wild-type sequence,
which may affect various conformational constraints
asso-ciated with active site flexibility
The aim of the present study was to further explore the
local flexibility concept in the adaptation strategies of
enzymes to low temperatures As in the previous study [16], our interest is focused to the vicinity of the active site of the psychrophilic alkaline phosphatase from the Antarctic
Table 1 Enzymatic and thermodynamic parameters of the psychrophilic alkaline phosphatase and mutants Reported values were determined at
10 C The k cat values were calculated from V max using a molecular weight for the enzyme of 76122 Da in a buffer containing 1 M diethanolamine-HCl pH 10, 10% glycerol, 10 m M MgCl 2 , 1 m M ZnCl 2 , and 10 m M pNPP E a values were calculated from the slope of the Arrhenius plots in the temperature range 5–25 C for native and G261A/Y269A mutant, and 5–15 C for the G261A mutant Thermodynamic parameters DG #
, DH#, TDS # were calculated as described previously [27].
Enzyme
k cat
(s)1)
E a
(kJÆmol)1)
DG# (kJÆmol)1)
DH# (kJÆmol)1)
TDS# (kJÆmol)1)
D(DG#) n-m
(kJÆmol)1)
D(DH#) n-m
(kJÆmol)1)
TD(DS#) n-m
(kJÆmol)1) Native 1212 42.8 52.48 40.45 )12.03
G261A 423 106.5 54.96 104.15 49.19 )2.48 )63.7 )61.22 G261A/Y269A 310 45.1 55.69 42.75 )12.94 )3.21 )2.3 0.91
Fig 3 Kinetic studies of wild-type and mutant alkaline phosphatases.
(A) Temperature dependence of k cat of TAB5 (d), mutants G261A
(r), G261A/Y269A (j) and E coli (·) alkaline phosphatases at
temperature range 5–25 C k cat values were determined in a buffer
containing 1 M diethanolamine-HCl pH 10, 10% glycerol, 10 m M
MgCl 2 , 1 m M ZnCl 2 , and 10 m M pNPP at 20 C Alkaline
phospha-tase activity was followed spectrophotometrically utilizing
nitro-phenyl phosphate (pNPP) as substrate The release of product,
p-nitrophenolate, was monitored by measuring the absorbance at
405 nm using a PerkinElmer photometer Reported values are the
average of three measurements The standard deviations do not exceed
10% (B) Arrhenius plots of TAB5, mutants G261A,G261A/Y269A
and E coli alkaline phosphatases Symbols are as in (A) Reported
values are the average of three measurements The standard deviations
do not exceed 10% (C) Thermal inactivation profiles of E coli and
TAB5 alkaline phosphatases Enzymes were incubated at 50 C, in a
buffer containing 1 M diethanolamine pH 10.0, 10 m M MgCl 2 , 1 m M
ZnCl 2 and 10% glycerol for different time periods and they were
subsequently incubated on ice for 30 min The remaining activity was
measured at 20 C Symbols are as in (A) Reported values are the
average of at least two measurements The standard deviations do not
exceed 10%.
Trang 5strain TAB5 We particularly attempted to investigate the
functional importance of the Gly pair, located in the vicinity
of the active site of the cold adapted enzyme and to study its
potential role in the establishment of its psychrophilic
character
This work uses, in accordance with more or less generally
established concepts, the energy of activation, Ea, as the
main criterion for the evaluation of the psychrophilic nature
of enzyme variants In cold adapted enzymes, this
param-eter generally tends to be lower compared to their
mesophilic counterparts [27] Furthermore, as a measure
of enzyme stability, thermal inactivation at 50C is used
We refer to stability in an activity sense and not in a
thermodynamic sense We therefore assume that even low
enzymatic activity is associated with a mutant that retains to
a considerable extent the overall fold of the wild-type
protein and that loss of activity is associated either with
perturbation of the native structure or local disruption of
the metal binding or the active site
The point mutation of Gly262fi Ala results in an
enzyme that exhibits very low activity (less than 1 : 1000
of the native enzyme) This fact did not allow the study
of its kinetic parameters and its thermal inactivation
profile However, this mutation demonstrates that at
position 262 the presence of Gly is essential, and a
mutation altering this residue results in a practically
inactive enzyme This Gly may provide the necessary
flexibility required for catalysis Several alkaline
phos-phatases have one Gly at the corresponding positions
261 and 262, while the psychrophilic enzyme has both
positions occupied by Gly
The most striking effect of the Gly261fi Ala
substitu-tion (Fig 2B) is the loss of the psychrophilic character as
deduced from the drastically altered Ea value (Fig 3B,
Table 1) As shown in Table 1, this is mainly attributed to
the considerable increase of DH#of the mutant compared
to the native enzyme This observation is in agreement with
previous reports [27], suggesting that the main adaptation of
psychrophilic enzymes lies in a significant decrease of DH#
with an unavoidable concurrent decrease of TDS# T he
slope of the Arrhenius plot, in the temperature range
5–15C, corresponds to an approximately threefold
increase of the Eavalue compared to the wild-type enzyme
Interestingly, while this mutant exhibits a considerable
decreased value of kcatat lower temperatures, at elevated
temperature (25C) the value of the same parameter
slightly exceeds that of the wild type (Fig 3A) This can
be also observed as a bend on the Arrhenius plot occurring
at temperatures > 20C, indicating that the Eavalue in this
temperature range is considerably lowered On the basis of
the model, the behavior of the G261A variant can be
interpreted in terms of constraints introduced by the Ala
side chain The presence of the additional Gly at position
261 possibly offers increased flexibility to the adjacent
residue Trp260 that forms part of the active site and
therefore facilitates the catalysis at low temperatures
Consequently, when the mutant G261A is driven to operate
in a cold environment, and the lack of Gly261 does not
allow the reaction to proceed as efficiently as in the case of
the native enzyme At higher temperatures, the additional
energy provided by the environment is sufficient and the
mutant can proceed with the catalysis as efficiently as the
wild type (Fig 3A) Investigation of the three-dimensional
homology-based model of the enzyme revealed that the methyl group of Ala261 side-chain could produce steric clashes with the aromatic ring of Tyr269, and these unfavorable interactions could lead to a decrease of local flexibility and an increased Eavalue
The validity of the above interpretation was further reinforced by the construction of the double mutant G261A/Y269A The additional substitution of Tyr269fi Ala was designed with the aim of reducing the spatial constraints originating from the side-chain interactions between Tyr269 and Ala261 (Fig 2C) The main difference between the G261A and G261A/Y269A enzymes is the restoration of the psychrophilic character in the double mutant Both mutations resulted in an enzyme exhibiting a significantly lower Ea, DH#and TDS#values similar to that
of the wild-type enzyme (Fig 3B, Table 1) In addition, considerable stabilization of the double mutant as compared
to the wild-type enzyme was observed (Fig 3C) This is probably the result of the relaxation of the side-chain packing constraints between positions 269 and 261 This explanation is additionally supported by sequence compar-isons As shown in Fig 1, in other alkaline phosphatases the corresponding residue at position 269 is often occupied by residues with smaller side chain when a larger than Gly residue is found at position 261 This is more striking in the case of the enzyme from the thermophilic alkaline phos-phatase from Thermotonga maritima where the presence of
a large side chain (Glu) at corresponding position 261 is accompanied by a Gly at corresponding position 269 thus compensating this increase in the side chain volume The contribution of Gly clusters in the cold adaptation of enzymes was also examined in the case of the mammalian psychrotolerant hormone-sensitive lipase [19] In that study,
a Gly rich loop (HGGG motif), which was only found in that enzyme, was extensively mutated and the activity of the engineered catalysts was analyzed in various temperatures However, it was found that although the HGGG loop was a critical structural element for the catalytic efficiency of the enzyme, the cold adaptation of the enzyme could not be attributed to the presence of the Gly cluster in this element The present study supports the idea that the Gly cluster,
in combination with its structural environment, is an essential feature of the psychrophilic character of TAB5 alkaline phosphatase It seems that the volume of the side-chains at positions 261 and 269 controls the psychrophilic character as judged from the levels of the Ea In the G261A mutant, this volume is increased (Fig 2B) and the enzyme proves to be as efficient as the native at elevated but not at lower temperatures The presence of Gly residues at both positions 261 and 262 is necessary for the enhanced specific activity of the enzyme in its natural environment; catalysts harboring a Glyfi Ala mutation in any of these positions exhibit a significantly decreased specific activity (Fig 3A) Consequently, the Gly cluster at this position plays a dual role, contributing both to higher catalytic efficiency and lower Ea
Moreover, the present work provides evidence that mutations introduced to Gly cluster produced enzymes that still exhibit psychrophilic properties while suitable compen-sating mutations may even produce mutants with increased stability To our knowledge, the present study along with a previous one from our laboratory describing the mutagen-esis of residues Trp260 and His135 of the same enzyme, are
Trang 6the first examples where rational redesign of residues, at or
close to the active site, has been used to demonstrate that the
psychrophilic character of an enzyme can be strongly
affected by very slight variations of its amino-acid sequence
Crystallographic studies of the mutants, aiming to further
test the hypotheses about the structural basis of kinetic
findings, are in progress
A C K N O W L E D G E M E N T
This work was supported by the TMR Network FMRX-CT97-0131.
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