Báo cáo khoa học: Implication for buried polar contacts and ion pairs in hyperthermostable enzymes pot

The three representative enzymes, AT, BG, and AM were selected due to the numerous structural information and stability data Keywords accessible surface area; buried polar contact; hyper

Implication for buried polar contacts and ion pairs in

hyperthermostable enzymes

Ikuo Matsui and Kazuaki Harata

Biological Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan

Introduction

Hyperthermophiles grow optimally at temperatures of

80–110C [1] Only represented by bacterial and

archaeal species, these organisms have been isolated

from all types of terristerial and marine hot

environ-ments Some of the enzymes from hyperthermophiles

are active at temperatures as high as 110C and above

[2] Recently, much effort has been directed towards

the isolation and characterization of enzymes from

hyperthermophilic archaea Interest in these enzymes

has increased because of their potential

biotechnolo-gical applications [3,4] and because of the need for a

better understanding of their intrinstic heating and denaturing resistance Elucidating the stabilizing mechanisms has been one of the greatest challenges in biochemistry and biotechnology [3–5]

This minireview encompasses the molecular determi-nants of protein stability, and compares the various molecular structures of amino acid aminotransferase (AT), b-glycosidases (BG), and a-amylases (AM), from different origins, including hyperthermophiles, thermo-philes living at around 65–75C, and mesophiles living

at room temperature The three representative enzymes, AT, BG, and AM were selected due to the numerous structural information and stability data

Keywords

accessible surface area; buried polar

contact; hyperthermophilic archaea;

hyperthermostable enzyme; ion pair;

molecular structure; Pyrococcus; subunit

interaction; thermostability

Correspondence

I Matsui, Biological Information Research

Center, National Institute of Advanced

Industrial Science and Technology (AIST),

Tsukuba, Ibaraki 305, Japan

Fax: +81 29 8616151

Tel: +81 29 8616142

E-mail: ik-matsui@aist.go.jp

(Received 28 February 2007, accepted

17 May 2007)

doi:10.1111/j.1742-4658.2007.05956.x

Understanding the structural basis of thermostability and thermoactivity, and their interdependence, is central to the successful future exploitation of extremophilic enzymes in biotechnology However, the structural basis of thermostability is still not fully characterized Ionizable residues play essen-tial roles in proteins, modulating protein stability, folding and function The dominant roles of the buried polar contacts and ion pairs have been reviewed by distinguishing between the inside polar contacts and the total intramolecular polar contacts, and by evaluating their contribution as molecular determinants for protein stability using various protein structures from hyperthermophiles, thermophiles and mesophilic organisms The anal-ysis revealed that the remarkably increased number of internal polar con-tacts in a monomeric structure probably play a central role in enhancing the melting temperature value up to 120C for hyperthermophilic enzymes from the genus Pyrococcus These results provide a promising contribution for improving the thermostability of enzymes by modulating buried polar contacts and ion pairs

Abbreviations

available from widely distributing origins, including

hyperthermophiles, thermophiles and mesophiles

Background: there is no single

dominating factor for the

thermostability of proteins

Many crystal structures of hyperthermophilic enzymes

have been reported, and several factors responsible for

their extreme thermostability have been suggested It

was proposed that the stability of a protein could be

increased by selected amino acid substitutions that

decrease the configurational entropy of unfolding [6]

Proline reduces the flexibility of the polypeptide chain

The mutations, Glyfi Xaa or Xaa fi Pro should

decrease the entropy of a protein’s unfolded state and

stabilize the protein A number of the thermophilic

and hyperthermophilic proteins also use this

stabiliza-tion mechanism [1] The stabilizing role of a large and

more hydrophobic core was proposed based on

experi-mental evidence obtained using chimeric

methanocco-cal adenylate kinases [7] The stability properties of the

chimera constructed from the Methanococcus voltae

and Methanococcus jannaschii adenylate kinases

indi-cated that cooperative interaction within the

hydro-phobic protein core plays an integral role in increasing

the thermalstability Another potential stabilizing

fac-tor is the high packing density of the molecule A

com-parison of the hyperthermophilic aldehyde ferredoxin

oxidoreductase with the mesophilic aldehyde

ferre-doxin oxidoreductase revealed that the former has a

small solvent-exposed surface area [8] A comparison

of citrate synthases from a hyperthermophile

(Pyrococcus furiosus), thermophile (Thermoplasma

acidophilum) and mesophilic organism (pig) indicated

that increased compactness of the enzyme might be one

of the major factors required for its thermostability [9]

An increase in the number of ion pairs and

hydro-gen bonds is also important for thermostability The

crystal structure of glyceroaldehyde-3-phosphate

dehy-drogenase from a thermophile, Thermus aquaticus, has

been compared with that of three

glyceroaldehyde-3-phosphate dehydrogenases of different origins [10]

From the results, a strong correlation between

thermo-stability and the number of hydrogen bonds between

charged side chains and neutral partners was found

There are two reasons why proteins may use

charged-neutral hydrogen bonds rather than salt links or

neu-tral-neutral hydrogen bonds to stabilize protein [10]

First, the desolvation penalty associated with burying

charged-neutral hydrogen bonds would be less than

that of burying ion pairs because of only one charged

residue being involved Second, the enthalpic reward

of charged-neutral hydrogen bonds is greater than that

of neutral-neutral hydrogen bonds because of the charge–dipole interaction By a structure comparison

of [Fe3S4]-feredoxin from the hyperthermophilic archa-eon P furiosus with those from the thermophile and mesophile, further significant roles of the hydrogen bonds on the hyperthermostablity have been reported [11–13] The P furiosus feredoxin structure shows a higher degree of hydrogen-bond network than other homologus ferredoxins, and this is believed to be the main reason for the observed increased thermostability with a denaturation temperature of over 100 C

An increase in the number of ion pairs, especially

in networks, is observed nearly in every hyperthermo-stable protein [1,14] The structral comparison of an

O6-methylguanine-DNA methyltransferase from a hyperthermophilic archaeon, Thermococcus kodakara-ensis, with the mesophilic counterpart from

Escherichi-a coli suggested that four additional buried ion pairs between a-helices might play a key role in its stabiliz-ing mechanism [15] The amino acid residues formstabiliz-ing interhelix ion pairs are buried relatively more often than those of intrahelix ion pairs because the aver-aged solvent-accesible surface areas of amino acid res-idues forming inter- and intrahelix ion pairs are 39.5 A˚2 and 98.9 A˚2 per residue, respectively This suggests the internal location of interhelix ion pairs in the molecule The interhelix ion pairs in the interior

of the protein presumably enhance the stability of the internal packing (tertiary structure) Furthermore, a stabilizing function has also been proposed for buried ion pairs in Thermosphaera aggregans BG (BGTa) [16] In four different sequences of hyperthermophilic BGs (BGTa from T aggregans, BGSs from Sulfolobus solfataricus, BGPf and b-mannosidase from P furio-sus), 28% of the residues are strictly conserved In the aligned sequences, a strict conservation is observed among the residues participating in forming internal ion pairs; however, only 26% of the surface ion pairs are conserved, consistent with the average sequence conservation among these sequences The homohexa-meric structure of hyperthermophilic glutamate dehy-drogenase (GluDHPf) (t1⁄ 2, 12 h; 100C) from

P furiosus was compared with the mesophilic GluDHCs (t1⁄ 2, 30 min; 52C) from Clostridium sym-biosum The comparison revealed that the hyperther-mostable enzyme contains a striking series of ion pair networks on the surface of the protein subunits, and partially buried at intersubunit and inter-domain interfaces, not found in the mesophilic counterparts [1,17–19] The importance of intersubunit ion pairs to the structural stability of GluDHTk from a hyperther-mophile, Thermococcus kodakaraensis, was examined

by site-directed mutagenesis, involving systematic

addition or removal of ion pairs [20] These results

proved the important role for the intersubunit ion

pairs in stabilizing the GluDHTk molecule However,

completely different results were reported from a

structural analysis of GluDHPi from a

hyperthermo-phile, Pyrobaculum islandicum [21] The number of

in-tersubunit ion pairs in the homohexameric GluDHPi

molecule was much smaller than that in GluDHPf or

the mesophilic GluDHCs These findings suggest that

the major molecular strategy for thermostability may

differ for each hyperthermophilic enzyme [21] The

significant role of the entropic effect, due to shorter

surface loops, on the thermostability of tryptophan

synthase a-subunit from P furiosus (a-subunit-Pf) was

reported [22] The thermostability of the a-subunit-Pf

molecule was examined by differential scanning

calo-rimetry (DSC) and by comparing the molecular

struc-ture with that of a mesophilic a-subunit-St molecule

from Salmonella typhimurium The DSC data indicated

that the greater stability of the a-subunit-Pf molecule

was not caused by an enthalpic factor From these

results, it was concluded that hydrophobic interactions

in the protein interior do not contribute to the higher

stability of the a-subunit-Pf molecule The increased

number of ion pairs, smaller cavity volume, and

entro-pic effects due to a shorter polypeptide chains, are

important in the hyperthermostability of the

a-subunit-Pfmolecule

A comparative analysis of the proteins

Thermococ-cus kodakaraensisKOD ribulose-bisphosphate

carboxy-lase⁄ oxygenase [23], Thermotoga maritima dihydrofolate

reductase [24] and phosphoribosylanthranilate

isomer-ase [25], and Aeropyrum pernix

2-deoxy-d-ribose-5-phosphate aldolase (DERA) [26] suggested that

oligomerization of subunits appears to be the factor

responsible for the hyperthermostability The area of

the subunit–subunit interface in the dimer of the

A pernixDERA is much larger compared with that of

the E coli enzyme Furthermore, the A pernix DERA

has an additional N-terminal helix that induces the

formation of a characteristic dimer–dimer interface

These results suggest that the hyperthermostability of

the A pernix DERA could be enhanced by the

forma-tion of a unique tetrameric structure unlike the dimeric

structure of the mesophilic counterparts (i.e the E coli

enzyme) [26]

Hence, protein stability appears to be attributable

to a combination of factors, which are related to

each other and their contribution to vary depending

on the proteins It is proposed that there is no single

dominating factor for the thermostability of proteins

[14]

Evaluating buried polar contacts and ion pairs as structural elements related

to the thermal stability

Because intermolecular and intramolecular polar inter-actions such as hydrogen bonds [11–13] and salt link-ages [1,14–22], appeared to be major factors that are responsible for hyperthermostablility, interatomic con-tacts involving main chain peptide groups and polar side chain groups of Asp, Glu, Arg, Lys, Asn, Gln, His, Thr and Ser were counted and classified on their location inside or outside of the molecule The number

of these contacts was divided by the total number of polar contacts and the results was used to evaluate the rigidity of the core region and the hydrophilic property

of the molecular surface For oligomeric proteins, in-termolecular polar contacts between subunits were also calculated The solvent-accessible surface area was cal-culated by the Lee and Richards algorithm (probe radius 1.6 A˚) [27] Next, the accessible surface area of

a protein molecule was divided by the number of amino acid residues and used as an indicator to com-pare the compactness of the protein structure

Structural elements responsible for thermostability: increase in molecular compactness, hydrophilicity of the molecular surface, and buried polar contacts

ATs have been widely applied to the large-scale bio-synthesis of unnatural amino acids, which are in increasing demand by the pharmaceutical industry for peptidomimic and other single-enantiomer drugs [28]

An aspartate aminotransferase gene homolog (ORF: PH1371) was identified by sequencing the genome of

a hyperthermophilic archaeon, Pyrococcus horikoshii [29,30] The gene (ArATPh) was expressed in E coli, and the product was purified to homogeneity The enzyme ArATPh was proven to be an aromatic amino-transferase [31] ArATPh is one of the most thermosta-ble aminotransferases reported to date far, with a melting temperature (Tm) of 120C The crystal struc-ture of ArATPh was determined at a resolution of 2.1 A˚ [31] and shown in Fig 1 as protein databank accession code (PDB ID): 1dju ArATPh has a homo-dimeric structure in which each subunit has two domains similar to other aminotransferases As shown

in Fig 1, the ArATPh structure is more compact due to shortened loops (colored in green) compared to those observed in thermophilic (Thermus thermophilus, PDB ID: 1bjw) and mesophilic (E coli, PDB ID: 1ars) According to the thermodynamic database of proteins

and mutants (ProTherm; http://gibk26.bse.kyutech.

ac.jp/jouhou/protherm/protherm.html) [32], the highest

Tm of an enzyme measured directly by DSC was

121.6C for cytochrome c3 from Desulfovibrio vulgaris

[33], although the Tmvalue of PhCutA1 from P

hori-koshiiwas reported recently to be 150C [34]

From the ArATPh structure, the accumulated

acces-sible surface area and intermolecular polar contacts at

distances shorter than 3.3 A˚ were calculated (Table 1)

Inside–inside contacts refer to amino acid residues that

are buried inside the molecule and they are not

sol-vent accessible, whereas surface–surface contacts refer

to residues that are exposed to the solvent, even

par-tially Such parameters that measure structure features

related to thermal stability were calculated for eight

aminotransferase molecules derived from

hyperthermo-philes, thermophiles and mesohyperthermo-philes, including

mam-mals such as pig and human [31,35–41], and are

summarized in Table 2 The optimal growing

tempera-ture of each living organism, the enzyme name, the

PDB ID, and the Tm measured by DSC are also

shown in Table 2 The accessible surface area divided

by the total residue number of the dimer was used as a

reference to evaluate molecular compactness The

occupancy of charged residues in the solvent-accessible area was useful to evaluate the hydrophilicity of the molecular surface

All aminotransferases listed in Table 2 are homodimers Their Z score and rmsd values range between 14.8 and 7.0 A˚ and between 1.07 and 2.38 A˚,

Fig 1 The crystal structures of ATs and

BGs The figures were produced using the

the hyperthermophilic ArATPh dimer

(PDB ID: 1dju), thermophilic AT dimer (from

Thermus thermophilus, PDB ID: 1bjw), and

mesophilic AT dimer (from Escherichia coli,

PDB ID: 1ars) a-Helices, b-sheets and loops

are colored in pink, blue and green,

respec-tively The cofactors, pyridoxal 5¢-phosphate

(PLP) molecules covalently binding to the

essential Lys residue, are shown with a

the hyperthermophilic BGPh molecule

(PDB ID: 1vff) and mesophilic BG (from

Paenibacillus polymyxa, PDB ID: 1bga) The

model is viewed along the axis of the barrel.

a-Helices, b-sheets and loops are colored in

pink, blue and green, respectively.

Table 1 The solvent-accessible surface area and intramolecular

amino-transferase (ArATPh) as a dimer from an hyperthermophilic archa-eon, Pyrococcus horikoshii.

Total accessible

Total buried residues

Total

The number and ratio of intramolecular polar

Inside–

inside

Inside–

surface

Surface–

surface

Subunit– subunit

a

Total residue number consisting of dimer excluding the disordered

Optimally growing temperature (

PDB ID

Melting temperature by

Accessible surface area

Occupancy of

surface area

Homology (%)

Z score

rmsd (A

Inside– inside (%) Surface– surface (%) Subunit– subunit (%) Nonsurface ionic (%)

Aromatic amino

Aspartate aminotransferase

Aspartate aminotransferase

Aromatic amino

Tyrosine aminotransferase

Kynurenine aminotransferase

respectively, reflecting a high structural similarity to

ArATPh, although the sequence similarity varied

between 43% and 17% The Tm of the mesophilic

ATEc from E coli measured directly by DSC is

approximately 63C [42], whereas that of the

hyper-thermophilic ArATPh is 120C [31] A comparison of

the accessible surface area per amino acid residue for

the enzymes (Table 2) shows that the value of ArATPh

is the lowest (28.0 A˚2), suggesting tightest molecular

packing Another prominent feature of ArATPh is the

largest occupancy of charged residues (Asp, Glu, Lys,

and Arg) at its surface (up to 73.3%), indicating a

hydrophilic molecular surface Moreover, the

fre-quency of buried polar contacts among all polar

con-tacts in distances less than 3.3 A˚ in the ArATPh

molecule is highest (18.3%) as shown in Tables 1 and

2 By contrast, the frequency of polar contact on the

surface of ArATPh is the lowest (36.0%) relative to

that of the other enzymes listed in Tables 1 and 2

Interestingly, the presence of polar contact at the

inter-face between the monomers is essentially the same for

all enzymes tested With an increase in the optimum

growing temperature of each organism, the molecular

compactness, surface hydrophilicity and ratio of buried

polar contacts of each aminotransferase appears to be

increased

BGs are a group of enzymes that hydrolyze the

b-glycosidic linkage between carbohydrates or between

a carbohydrate and a noncarbohydrate moiety The

BGs from the hyperthermophilic archaeon P

horiko-shii(BGPh) were crystallized in the presence of a

neu-tral surfactant, and the crystal structure was solved at

2.5 A˚ resolution [43] (Fig 1) Notably, the major

dif-ference of the amino acid sequence among BGPh,

BGTa from T aggregans [16], and BGSs from

S solfataricus [44], is the deletion of more than 50

residues from the BGPh sequence that are assigned to

loops As shown in Fig 1, the overall structure of the

hyperthermophilic BGPh (PDB ID: 1vff) looks very

similar to that of mesophilic BGs from Paenibacillus

polymyxa (BGPp) (PDB ID: 1bga) BGPh is a

mem-brane-bound enzyme that is extremely thermostable

and it has been shown to have high affinity for

alkyl-b-glycosides Hence, this enzyme may be used in

industrial applications for the degradation of sugar

derivatives and in the synthesis of various

alkyl-glyco-sides via transglycosidation or ‘reverse hydrolysis’ [45]

Parameters relevant to thermal stability were

calcu-lated for six BG molecules derived from

hyperthermo-philes, mesophiles and plants, and are summarized in

Table 3 [43–49] The oligomeric state of the BG

mole-cules listed in Table 3 varies from monomer (BGPh) to

homooctamer (BGPp); however, their Z score and

rmsd values range between 15.3 and 12.4 A˚ and between 1.47 and 1.66 A˚, respectively The sequence similarity varied between 35% and 30% These data show that there is a high structural similarity between

BG molecules; the main difference among them being their oligomeric state BGPh is more thermostable than the mesophilic BGPp molecule; t1⁄ 2 of BGPh at

90C is 15 h [45], whereas t1 ⁄ 2 of BGPp at 35C has been reported to be 15 min [46] The prevalence of charged residues at the surface of hyperthermophilic

BG is higher than that of the homologus proteins from mesophilic organisms (BGPh; 56.2%, BGTa; 52.1%); this suggests that the hyperthermophilic enzyme has a more hydrophilic surface The frequency of buried polar contacts in the BGPh molecule is the highest (14.4%) as shown in Table 3, whereas the accessible surface area per amino acid residue showed no promi-nent difference between hyperthermophilic and meso-philic BGs The molecular compactness of BGs was calculated for the monomeric form regardless of the oligomeric state, which varied from monomer to homooctamer: with increasing thermostability, the sur-face hydrophilicity and the percentage of buried polar contacts of the BG enzymes was also increased AMs catalyze the hydrolysis of a-(1,4)glycosidic linkages of starch components, glycogen and various oligosaccharides, and is an important industrial enzyme The a-amylase (AMPw) from the hyper-thermophilic archaeon Pyrococcus woesei, which grows optimally at 100–103C, was crystallized, and the molecular structure was solved at 2.0 A˚ resolution [50] Many a-amylases have been isolated and charac-terized from hyperthermophiles to mesophilic organ-isms [50–58] The AMs listed in Table 4 are monomers with known crystal structures Except for a distant relative of a-amylase, the aminomaltase from

T aquaticus, the Z score and rmsd values of the other AMs range between 13.3 and 10.0 A˚ and between 1.41 and 2.52 A˚, respectively, representing fairly good structural similarity to AMPw The sequence similar-ity of these AMs varied between 33% and 18% (aminomaltase was excluded from the comparison) The Tm value of AMPw measured by DSC is 112C [32], whereas the Tm values of mesophilic a-amylases from a fungi, Aspergillus oryzae, and from human were reported to be 62C and 67.4 C, respectively [32] A significant difference was observed in the fre-quency of internal polar contacts of the AM mole-cules; the value of the AMPw molecule is the highest (17.1%) among the AMs listed in Table 4 Furthermore, with an increase in thermostability, the ratio of buried polar contacts also increased How-ever, the molecular compactness, surface hydrophilicity

Optimally growing temperature (

(oligomeric state)

PDB ID

Half-life t 1

Accessible surface area

Occupancy of

surface area

Homology (%)

Z score

rmsd (A

Inside– inside (%) Surface– surface (%) Nonsurface ionic (%)

b (monomer)

b (homotetramer)

Thermosphera aggregans

b (homotetramer)

b (homooctamer)

Plant (Sinapis

Myrosinase (homodimer)

Plant (Trifolium

Cyanigenic b (homodimer)

Optimally growing temperature (

PDB ID

Melting temperature by

Accessible surface area

Occupancy of

surface area

Homology (%)

Z score

rmsd (A

Inside– inside (%) Surface– surface (%) Nonsurface ionic (%)

Bacillus stearothermophilus

t 1

Alkalophilic Bacullus

G6

and percentage of polar contact on the surface did

not show a significant trend from the mesophilic to

the hyperthermophilic AM (Table 4)

Conclusion

As shown in Tables 2–4, the most thermostable

enzymes from Pyrococcus species (e.g ArATPh,

BGPh, and AMPw) belonging to three different

pro-tein families, have the highest rate of buried polar

con-tacts compared to that of their mosophilic and

thermophilic counterparts In addition, ArATPh has a

much higher rate of buried ion pairs than ATs from

other species Recent surveys on the exposure of

ioniz-able groups to solvent [59], ion pairs [60], and the

des-olvation energy of these residues [61] using the protein

structure database, show that more than 30% of the

ionizable residues are fully or partially buried and

ion-ized in the internal of the molecule [62] Buried ionion-ized

residues appear to be more conserved than those on

the surface [62] Here, the dominant roles of the buried

polar contacts and ion pairs were reviewed by

distin-guishing between the inside polar contacts and the

total intramolecular polar contacts, and by evaluating

their contribution as molecular determinants for

pro-tein stability using various propro-tein structures from

hyperthermophiles, thermophiles and mesophilic

organisms Although more abundant data for the

structure⁄ stability relationships of various proteins

other than the representatives, AT, BG and AM, if

available, should be considered, the results reported

suggest strategies for improving the thermostability of

enzymes by modulating the internal polar contacts and

ion pairs

Acknowledgements

We thank Hideshi Yokoyama at University of

Shi-zuoka, School of Pharmaceutical Science, and Eriko

Matsui for their valuable advice and discussion

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