放線菌Streptomyces olivaceoviridis E-86由来ファミリー10キシラナーゼの触媒ドメイン中に存在するN末端およびC

末端αヘリックスの酵素安定性における重要性

誌名誌名 Journal of applied glycoscience

ISSNISSN 13447882

著者著者

金子, 哲伊藤, 茂泰藤本, 瑞久野, 敦一ノ瀬, 仁美岩松, 新之輔長谷川, 典巳

巻/号巻/号 56巻3号

掲載ページ掲載ページ p. 165-171

発行年月発行年月 2009年7月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

165

よAppl.Glycosci.， 56，165-171 (2009) @ 2009 The Japan巴seSociety of Applied Glycoscience

Regular Paper J主J~AQ

Importance of Interactions of the α・Helicesin the Catalytic Domain

N嗣 andC-Terminals of the Family 10 Xylanase from

Streptomyces olivaceoviridis E・86to the Stability of the Enzyme *

(Receiv巴dJanuary 26， 2009; Accepted March 24， 2009)

Satoshi Kaneko， 1，* * Shigeyasu Ito/ Zui Fujimoto，3 Atsushi Kuno，2 Hitomi Ichinose，1

Shinnosuke Iwamatsu2 and Tsunemi Hasegawa

2

1Food Biotechnology Division， Nationα1 Food Research Institute (2-1-/2， Kannondai， Tsukuba 305-8642， Japan)

2Department of Material & Biological Chemistry， Faculty of Science， Yamagata University (1-4-/2， Kojirakawa-machi， Yamagata 990-8560， Japan)

3Protein Research Unit， National Institute of Agrobiological Sciences (2-1-2， Kannondai， Tsukuba 305-8602， Japan)

Abstract: The intact crystal structure of family 10 xylanase (SoXynl0A) from Streptomyces olivaceoviridis in-dicates that the catalytic domain of SoXynl0A consists of nine α-helices (α0-8) and eight s-sheets (sI-8). In-teraction in the α幽helicesof N-terminal (α0) and C-terminal (α8) of catalytic domain of SoXynl0A by 3 hy-drogen bonds and 8 hydrophobic interactions is observed and predicted to playing an important role for the stability of the molecule. Therefore， the importance for the stability and folding of SoXynl0A were examined by using C-terminal truncated mutants of SoXynl0A. The thermostability was gradually decreased when the C-terminal was shortened; however， the enzyme activities were not influenced by the length of the C-terminal. The investigation of the stability using guanidine hydrochloride agreed with the expected results; namely the hydrophobic core was completed at Leu・300and the resulting stability was not changed if the C桐terminalwas longer than it. The thermostabilty slightly decreased when Asn-252 was replaced with Ala， suggesting hydro-gen bonding of Gly幽303with Asn-252 is also important for the stability of the molecule. When the effect of the interaction was observed in chimeric xylanases， which have a slight distortion in the structure， the C-terminal4 amino acids certainly increased the thermostability of the chimeric enzymes. However， the N-and C-terminal of CfXynl0A from Cellu/omonas fimi displayed by that of SoXynl0A decreased thermostability at the same degree as SoXynl0A， suggesting that the limit temperature of the interaction agrees with that of SoXynl0A and the distortion of both terminals make denaturating of the protein easy.

Key words: endo-s-l，4-xylanase， glycoside hydrolase family 10， thermostability， Streptomyces olivaceoviridis

The plant cell wall consists mainly of a complex mix-

ture of polysaccharides such as c巴llulose，hemicellulos巴

and p巴ctin.1)Xylan is the major component of the hemi-

celluloses found in plant cell walls.つrhebackbone of xy-

lan is formed byβ1，ふlinkedD-xylopyranose units to

which several side groups such asα-1，2-linked 4-0-methyl D-glucuronic acid and α寸，3-linked

レarabinofuranos巴 areattached.2)βXylanas巴 (EC3ユ1.8)

randomly hydrolyzes β1，4-g1ycosidic linkages within the xylan backbon巴 toyield short chain xylooligosaccharides

of varying length. Xylanases have many commercial uses，

such as in the paper manufacturing， animal fl巴ed，bread叩

making， juic巴andwine industries， and xylitol and xylooli-

gosaccharide production.3)

Thermostability is an important prop出 yof industrially

significant enzymes. Understanding the structural basis for

this attribute will underpin the future biotechnological ex-

ploitation of these biocatalysis. How巴ver，the term ther同

mostability does not refer to a well-defined physico-

* Importance of N-and C-Terminals Interaction in a Xylanase. 村 Correspondingauthor (Tel. +81-29-838-8063， Fax. +81-29 838-7996， E同mail:sakaneko@affrc.go.jp.).

ch巴micalproperty of a protein， but is generally used to in-

dicate the ability of some proteins to function at elevated

t巴mperaturesand to withstand them for some time. Such

an ability implies that the protein is able to remain folded

in its functional nativ巴 stateunder such conditions. Be回

cause many other factors b巴sidestemperature affect pro四

tein stability， thermostability at best can only be loosely

defined. A number of previous studies have shown that

various factors can increase protein thermostability， such

as an efficient packing of the hydrophobic core:-6) electro-

static interactions such as salt bridges and hydrog巴n

bonds，5川)and the stabilization of helix dipoles.9)

Streptomyces olivaceoviridis E叩 86produces both family 10 and 11 xylanases.叫 11) The family 10 xylanas巴

(SoXynlOA) has been well characterized.iO.12-22) SoXynlOA

consists of two domains， a catalytic domain (belonging to

GH family 10) and a carbohydrate-binding module

(CBM) (belonging to CB悶 family13)， both of which ar巴

connected by a Gly /Pro-rich linker region.10) The deletion

of the CBM of SoXynlOA resulted in about a 200

C d巴-

crease in the optimum temp巴ratureof SoXynlOA from

Streptomyces olivaceoviridis E-86 after removal of the C-terminal CBM.19

)

166 J. Appl. Glycosci.， Vol. 56， No. 3 (2009)

Generally， th巴 1・emovalof CBMs does not affect any of the othei properties of the enzymes except that of the hy-

drolytic capability toward insoluble substrates.16) However，

there are some reports in which the thermostability of

family 10 xylanas巴sis reduced after th巴 removalof じterminal CBMs.1刊 9)For example， the thermostability dis-

played by a family 10 xylanase from Streptomyces halste-dii was reduced wh巴nthe enzyme lost its C同 terminalCBM by proteolytic cleavage.17.18) Therefore， both the N-and Cぺ巴rminalregions of the CBM seem to play an im-portant role in th巴stabilityof th巴balTelmodule.

Herein， we report the importance of th巴 interactionsbe-tween the N-and C-terminal regions of the catalytic do田

main in determining th巴stabilityof the mol巴culeusing C同

terminal truncated mutants and chimeric xylanases.

MATERIALS AND METHODS

ConstJ・uctionand production of mu仰 ltenzymes. To

express SoXynlOA and its C-terminal truncat巴dmutants，

th巴 appropriateDNA sequences w巴r巴 amplifiedby the po-lymerase chain r巴action(Fig. 3) and the resulting frag-

ments were clon巴dinto the pQE60 vector (QIAG町、~ K. K.) as described pr巴viously.23)Each DNA fragment was

amplified using a set of primers which includ巴deith巴rth巴

NcoI or the BamHI restriction sites， enabling the PCR products to be cloned into NcoI/BamHI-r巴strictedpQE60.

pET28a/CfXyn10A was construct巴das described previ-ously.24) Th巴 constructionand expression of th巴 chimeric

xylanases such as FCF-C4 and FCF-C5 are d巴scribedin another pap巴r.25)CfXynlOA-AESTL and CfXynlOA-AETTL were constructed， as shown in Fig. 4， and， with the aid of th巴 signalsequence fr・omSoXynlOA， the巴n-

zymes were produced. The StuI site was introduced by an

inverse PCR right after the signal sequ巴nceof SoXynlOA and pQE60/SoXynlOA was used as a template， then it was digested with StuI and BamHI. Alternatively， the ap-

propriate CfXynlOA gene was amplified using a primer designed to encode the amino terminal and C-terminal of

SoXynlOA with the StuI and BamHI restriction sit巴s.pQE60/CfXynlOA-AESTL and pQE60/CfXynlOA向

AETTL were constructed by the adhesion of the appropri-ate fragm巴nts.For the production of the enzymes， a pET28a vector was employed as described in a pr巴VlOUS

report.24)

Circular dichroism spectr，ααnd steady state kinetic

studies. Th巴 circular dichroism (CD) spectra of

CfXynlOA， CfXynlOA悶 AETTLand CfXynlOA-AESTL were measured using conditions r巴portedpreviously.24.26)

Steady山 statekinetics were inv巴stigatedas previously re-ported;24.26) briefly the reaction mixture containing p-

nitrophenyl-存ひxylobioside(PNP-X2) at various concerト

trations in 259も (w/v)McIlvain巴 buffer(a mixture of 0.1 M citric acid and 0.2 M NaAP04， pH 7.0) containing 0.05% (w/v) bovine serum alburnin (BSA) was incubated

at 300

C for 5 rnin and then 50μL of enzyme solution was added. The amount of p-nitrophenol (PNP) r巴leasedwas determined by monitoring th巴 absorbanceat 400 nm with

a spectrophotometer (DU-7400; Beckman). PNP-X2 was synthesized by th巴 methoddescribed in a previous p仕

per.27) Th巴 xylobioseused in the synth巴siswas purified

from ‘Xylobiose Mixturザ (SuntoryHoldings LtdふMeasurement of enzyme activi，砂・ Each assay mixture

contained 250μL of a 2 mM PNP-X2 solution， 200 IlL of

0.2 M phosphate buffer， pH 5.7 and 50μL of巴nzymeso-lution. The reactions were calTied out at the specified tem-

peratures for 10 min whereupon they wer，巴 stoppedby the addition of 0.5 mL of a 0.2 M Na2C03 solution， and the

amount of PNP released was determined at 408 nm (re-f巴nedto hereafter as the standard method).

To determine the optimum temperatures of the various enzymes， the enzym巴 activitieswere measured at selected

temperatures. Each enzyme was adjusted to the same con-centration (0.05 U/mL at 30

oC) before use in th巴assay.

The stabilities of the enzym巴sin guanidine hydrochlo-ride were investigated as follows. The enzym巴 solution

(0.05 U/mL at 30o

C) was pre-treated with guanidine hy-drochlor対eat 25

0

C for 30 min. The residual enzyme ac-。

tivity was then measured at 30~C under・thesam巴 concen-tration of guanidine hydrochloride used for the pre回

tr巴atmentstep.

The thermostabilities of th巴 enzymesw巴r巴 determinedas follows. The enzym巴 solution(0.05 U/mL at 30

oC)

was p印刷incubatedat various temperatures for 2 h. The re-

sidual巴nzymeactivity was then measur‘巴dat 450

C.

RESULTS

Interaction of the N幽 αndC -terminals in the catalytic

domain of So均mIOA.

SoXynlOA consists of 436 amino acids.23) SoXynlOA

consists of a catalytic domain and a CBM， and a Gly-rich linker which couples the two modules.23) The crystal struc-

ture of intact SoXynlOA shows that the catalytic domain consists of nineα叩 helices(α0-8) and巴ights-sh巴巴ts(sl 8) and that the domain is completed at the end of α8， at residue Asn-301.20) The linker， starting at Gly-302 and firト

ishing at Ser-316， is followed by the family 13 CBM run-ning from Gly“317 through to Thr-436.20) As shown in

Fig. 1， interactions between theα-h巴licesof th巴 N-termma討1(αO的)and C-t巴erm

OぱfSoXynlOA are obs 巴伐rved.Hydrophobic interactions are formed by Leu-5， Ala-8 and Ala-9 in α0， Phe-16 in s1，

and Leu-282， Phe-283， Tyr-293， Vaト296，L巴U町 297and

Leu-300 inα8. Three hydrogen bonds， namely N82 and 081 of Asn-301 inα8 hydrogen bonding with 0 of Glu-2

and NE2 of Gln回 11in α0， respectively， and 011 of Tyr-293 hydrogen bonding with OE1 of Glu-37， cover the hy-drophobic core so that it does not appear on the outer sur-

fac巴 ofthe molecul巴. Th巴seinteractions ar巴 predictedto be important for the stability of the molecule. The cata-1ytic domain alone (residues 1-303， abbreviated as Cat

(303)) expr巴ssedin Escherichia coli shows the enzymatic activity; 19) th巴refore，w巴 construct巴dC-te口ninaltruncated

mutants (Cat (299)， residues 1-299; Cat (300)， residu巴s1-300; and Cat (301)， r巴sidues1-301) (Fig. 2) to investigat巴

the importanc巴 ofthese regions for th巴 thermostabilityof

白eenzyme.

Enzyme activi，砂 andstabili.砂ザ theC-terminal trun-

cated muωnts of So勾11l10A.

The kinetic parameters of the mutant enzymes for PNP-

Importance of N-and C-Terminals Interaction in a Xylanase 167

(A)

Fig. 1. Interaction of the N-and C-terminals of th巴 catalytic do-main of SoXynlOA

(A) Whole view of the crystal structure of SoXynlOA complexed with arabinofuranosylxylotriose (pdb code 1 v6v).") (B) Close view of the interface between th巴N-and C-t巴rminalsof the catalytic do-main. Hydrogen bonds are shown as dashed lines colored in light blue and the side chains of the amino acids forming th巴 hydropho-bic cor巴 areshown in orange. The N-terminal region including α-helices，αo and α1， is shown in pink and the C-terminal region fromαーh巴lixα8 is shown in green. Bound arabinofuranosyl-xylotriose was colored in blue

SoXyn10A

Cal剖yticdomain Carbohydrale binding module

¥Gly-rich linker

Cal (303) 303

Cal (301) 301

Cal (300) 300

Cal (299) 299

Fig. 2. Construction of C-terminal truncated mutants of SoXynl0A.

SoXynlOA consists of 436 amino acids comprising a catalytic domain， a CBM and a Gly-rich linker.'O) The intact crystal structure of SoXynlOA suggests the catalytic domain consists of 301 amino acids (1-301) and that the linker starts at Gly-302 and ends at Ser・316 foliowed by出巴 CBMwhich runs from Gly-317 to Thr-436.'O) The C-terminal truncated mutants were constructed by PCR accord-ing to the method described in a previous repo目20)

X2 are shown in Table 1. Th巴reare no significant differ-

巴ncesin eith巴rthe Km or kcat values of any of the en-

zymes， indicating that the deletion of the C-terminal of

th巴 enzymedid not affect enzym巴 activity.In the crystal

structure of SoXynlOA， the hydrophobic core of the cata-lytic domain is completed at L巴u-300and s巴veralhydro-

gen bonds covering the core prevent the hydrophobic

amino acids app巴紅ingthe outer surface of th巴 molecule

To confirm the hypothesis that th巴 hydrogenbonds help

to protect th巴hydrophobiccor巴， the stabilities of the mu-

Table 1. Activity of native and mutants of SoXynl0A and CfXynlOA against PNP-X，・

Enzyme Km (mM) kcat (S-I)

SoXynlOA 2.1 40 Cat (303) 2.1 42 Cat (299) 1.9 41 CfXynl0A 0.012 7.0 CfXyn1 OA-AETTL 0.012 8.2 CfXynlOA-AESTL 0.015 10.8

明 nalsequen四 Stu1

s珂H晦甲a即n悶、沼閣a剖l同s問u岨@問 e

↓Ir…PCR

↓SI叫 eslionn 1

↓ 担捌訓川Illi明g伊似a剖t

Slu 1

BamHI

kcat/Km (S-I/mM)

19 20 22

583 683 720

CfXyn10A A-πLKEAADQAGR 14 ----305 YAAVMEAF 312 SoXyn10A AES'礼GAAAAQSGR 14 ----293 YTAVLNALNGG 303 CfXyn10A-AETIL AETILKEAADQAGR 14 ----305 YAAVMEAFNGG 315 びXyn10A-AESTL AES'礼 臥AAAQSGR 14 ----305 YAAVlNALNGG 315

Fig.3. Construction of CfXynlOA-AESTL and CfXynl0A-AET寸L.

A pQE60 vector with the SoXynlOA signal sequence was con-structed using th巴 inversePCR method. The CfXynl0A gene was amplified by PCR， and it was introduced into the vector at the StuI/SamHI r巴stnctlOnsJte.

tants in guanidine hydrochlorid巴 weremeasured. The re-

sults of the stability measurements in guanidine hydro-

chloride are shown in Fig. 4 (A). The stabilities of al1 of

the enzymes tested， except that of Cat (299) which dis-

played a reduction in its stability relative to the other en-

zymes， w巴r巴 thealmost the same. In th巴 crystalstructure

of SoXynlOA， the hydrophobic core of the catalytic do-

main is completed at Leu-300 and several hydrogen bonds

covering th巴 coreprevent the hydrophobic amino acids

appearing the outer surface of the molecul巴. The guani

din巴 hydrochloride weakens both the hydrophobic and

electrostatic interactions. The differenc巴 inthe stability

seems to be observed when the key interaction stabilizing

the SoXynlOA molecule is broken. That is to say， Leu-

300 which completes the hydrophobic core of N-and C-

terminals， greatly contributes to the stability of the mole-

cule. This result suggests that the hydrophobic core in the

N-terminal-C-terminal int巴ractionis complete at residue

Leu-300. In contrast， the thermostability gradual1y de-

creased in relation to the decreasing length of the C-

terminal (Fig. 4 (B)). These denaturations of the enzymes

wer巴 non-reversible.The optimum temperatures of巴ach

of the mutants were 60， 55， 50 and 450

C for Cat (303)，

168

(A)1::

J. Appl. Glycosci.， Vol. 56， No. 3 (2009)

8日

# 70 妄 60

主羽詰 ω23日制

520 a: 10

0.5 1.5 2.5

Concn. 01 guanldlne hydrochlorlde (mMl

(8) 2

~. I d ] . [1

0 20 30 411 50 60 70 80

Tempera加re(・Cl

( C)100

# 80

さ60(.) ・6ω411 主M

e

~ 20

0 30 35 411 45 50 55 60 65 70

丁目np冒 a旬re(・Cl

Fig. 4. The stability in guanidin巴 hydrochlorid巴 (A)，thermostability (B) of the C.terminal truncated mutants of SoXynlOA and thermosta.

bility of the Cat (303).N252A mutant (C)

・，Cat(303); 0， Cat(303).N252A， T， SoXynlOA;圃， Cat(299); ....， Cat(300); +， Cat(301)

、~}

Fig. 5. lnteraction between Gly.303 and Asn.252 in the N-and C-terminal int巴ractionof Cat (303)

100

; / /

〆

IL--HM

n

u

n

u

n

o

n

u

n

m

U

F

O

-

a守

内

4

(Jo-E一〉=ueω〉

=20巴

0 30 35 40 45 50 55 60 65 70

¥ミ0 30 35 40 45 50 55 60 65 70

Tempera加re('・ClTemp冒 awre(・Cl

Fig. 6. The thermostability of chimeric xylanases and their C-terminal truncated mutants.

0， FCF-C4 (299);・，FCF-C4 (303);巴， FCF-C5 (299);・， FCF-C5 (303)

(A)100 90

80

~ 70 主 60

5ω 帽。40〉

宮 30

a 20 10

(8)20000

15000

10000

5000

一切00

。

Concn. 01 guanidlne hydr田 hlorlde(mMl

o I -10000 o 0.5 1.5 2 2.5 195 205 215 225 235 245

n町3

(C)2.5

2

~ 〉

31.5

0

21 ω E

0.5

0 30 40 50 60 70 80 90

T酎np冒 awre(・Cl

Fig. 7. The stability in guanidin巴 hydrochloride(A)， CD spectroscopy (B) and thermostability (C) of CfXynlOA， CfXynIOA-AESTL and CfXynIOA-AETTL.

X， CfXynIOA-AETTL;女，CfXynlOA;・， Cat (303); +， CfXynlOA-AESTL

Importance of N-and C-Terminals Int巴ractionin a Xylanas巴 169

Cat (301)， Cat (300) and Cat (299) respectiv巴ly.The ver-tical axis of Fig. 4 (B) shows that th巴 activityof each of

the enzymes is referenced to 1.0 at 30oC， reflecting the

specific activity of each of the enzymes at the various

temperatures. The activities of all of the enzymes up to 45

0

C are identical， indicating that the loss of enzyme ac-tivity directly reflects th巴 stabilityof the N-and C-

terminal interactions. To our surprise， the stability of Cat (303) and Cat (301) were not th巴 same，but that was ex-plained by the crystal structure of Cat (303). An addi-

tional hydrog巴nbond between Asrト252and Gly-303 was observed (Fig. 5) and it seem巴dto influence th巴 stabilityof th巴 molecul巴. To test the hypothesis， the N252A mu-

tant was constructed. The stability of the N252A mutant，

as shown in Fig. 4 (C)， supports the concept that the hy-

drogen bond between Asrト252and Gly-303 relates di-rectly to the stability of the catalytic domain. Th巴 numberof hydrogen bonds and hydrophobic int巴ractionsare di-

rectly r‘elated to the thermostability of the enzyme.

Effect of the interaction of the N闘 αndC-terminal re-gions of the cataちItiCdomains of the chimeric en・

zymes. It was evident from the investigations described above

that the 4 amino acids of the C-terminal of the catalytic domain (i.e. thos巴 involvedin the int巴ractionbetwe巴nthe

C-and N-terminals) were greatly involved in the stability of the enzyme. In order to examine whether th巴 interac-tion of the C-and N-terminal regions was also effectiv巴

in the chimeric xylanases， in which modules 4 or 5 were replaced by the CfXynlOA from C. funi producing a

slight distortion on the inside of their structur，巴 andaddi-tional strain in the folding of molecules，25) the th巴rmosta-

bilities of th巴 chimericxylanases were investigated. The r巴sultsare depicted in Fig. 6 and they indicate that it is

possible to make thermostable proteins even if the inside of the protein is distorted if both ends of the protein

molecule are still able to firmly interact with each other

Iny・'oductionof the interactions of the N-and ι terminals in CfXynl0A. Because the interaction of the C-and N-terminals of

SoXynlOA was effective for the stability of the molecule，

the interactions of SoXynlOA were introduc巴din th巴other family 10 xylanase. CfXynlOA from C. fimi was se-l巴ctedfor the target enzyme because the crystal structure of CfXynlOA has been reported28) and th巴 aminoacid se-

quence of N-and C-terminals of CfXynlOA is different

from those of SoXynlOA (Fig. 3). The introduction of the N-and C司 terminalint巴ractlOns

to CfXynlOA did not alter the observed kin巴ticparame-

t巴rsof the enzyme relative to the native CfXynlOA

(Table 1). The stability of th巴 chimericCfXynlOA eルzymes along with the native CfXynl0A are shown in Fig. 7. The stabilities of both chimeric CfXynlOA eルzymes in guanidine hydrochloride were almost th巴 same

as for the native CfXynlOA (Fig. 7 (A))， indicating that the replacement of both terminals by thos巴 ofSoXynlOA

did not affect the overall structure of th巴 enzyme.This is also supported by the CD spectra results (Fig. 7 (B)) and

the kinetic studies (Table 1). In contrast， the thermostabil-

ity of the CfXynlOA巴nzymedecreased to the sam巴 lev巴l

as that of Cat (303) (Fig. 7 (C)) in spite of the stabilities of CfXynlOA， CfXynlOA田AETTL and CfXynlOA-AESTL in guanidin巴 hydrochlorid巴 beingthe same. The observation of a decrease in the thermostability of

CfXynlOA mutants would indicate that the temperature 。

limit for the interaction is 60T (Fig. 7 (C)). The distor-

tion of both terminals makes it easier to denature th巴 pro-t巴in.This conesponds to the observation with the S. halstedii xylanas巴 inwhich XysVW， having an elongated linker region was less stable than XyslS.17.18)

DISCUSSION

Thermostability is one of the most impOltant factors of

enzymes that catalyze industrially relevant reactions. Therefor巴， understanding of the structural basis for ther-

mostability has attracted consid巴rableinter・巴st.In several reports， the structur巴 ofthe thermostable family 10 xyla同

nase was solved and the implications for the evolution of thermostability in family 10 xylanases and other (α/戸)8-barrel巴nzymeswere discussed.29，30) The structural analysis

of family 10 xylanases outlined in the papers suggests that

thermostability is acquired mainly by improved hydropho-bic packing/9，301 favorable int巴ractions of charged sidechains with helix dipoles，29.30) and the introduction of

proline residues at th巴 N町民rminusof helices.29) However，

th巴 authorsdo not refer to the most terminal helices of the

enzyme (e.g.α0) or the interaction of the N-and C四

terminals which we were able to examine in this study

and we hav巴 demonstratedthat the interactions of the N同

and C-t巴rminalregions of the catalytic domain play a

critical role in the stability of family 10 enzymes. Th巴 hypothesisthat th巴 N-and C-terminal regions of

family 10 xylanases are implicated in the thermostability of the enzyme has been suggested in several reports.叶 19.31)

A family 10 xylanase from S. halstedii JM8 has been r・'e-ported as consisting of an N-t巴rminalfamily 10 catalytic domain， a C回 terminalc巴llulose回bindingdomain and a Gly-rich linker conn巴ctingthe two domains.32) The recombi-

nant enzyme of this xylanase was proteolytically cleaved

in the middle of the Gly-rich linker when the gene was

expressed in S. lividans and the degraded enzyme (XyslS) was found to b巴 morestable than the wild type enzym巴 (XyslL).l7) In addition， the length of th巴 C-

t巴rminalwas increased and decreased relative to XyslS

and the stability of these enzymes were less than that of XyslS.18) The other evidence to support the above hy-

pothesis can be found in th巴 thermostabilizingdomain. This domain was first located in T. maritima XynA.3i) Re-

moval of this domain significantly reduced th巴 observedthermostability of the enzym巴31)The homologue of this

domain has been found in several xylanas巴sand finally it was d巴monstratedthat the th巴rmostabilizingdomain is ac-tually a xylan-binding domain;14.15) therefore it is apparent

that the t巴rminalof th巴 catalyticdomain is quite import昌nt

to the stabil

170 J. Appl. Glycosci.， Vol. 56， No. 3 (2009)

xylanases. Introducing a disulphide bond b巴tweenthe dis-

ordered、N and C termini of CmXynlOB from Cellvibrio mixtus confl巴rredan increase in thermostability.33.34) Wh巴n

the C-terrninal of the catalytic domain of S. lividans

XynA was elongated by four amino acids， X-ra~ crystal-

lography data was successfully obtained at 1.2 A resolu時

tion compared to the parental r・esolutionof 2.6 A.35)

The investigations using chimeric enzymes suggested

出atthe interaction is applicable in protein engineering be-

cause it is still possible to use the protein if it has some

degree of strain inside the structure. Attempts to increase

th巴thermostabi1ityof CfXynlOA were unsucc巴ssful;how剖

ever we have demonstrated the possibility of using these

interactions to change th巴 stabilityof the enzyme without

changing other properties such as substrate specificity.

The interaction of the N-and C-terminals is observ巴din

all barrel type enzymes. Although a thermostabilizing do-

main does not exist，14.15) this interaction is sti1l expected to

be able to be exploited in proteins with a thermostabiliz-

ing domain to change th巴 stabilityof TIM-barrel proteins.

In some proteins， the technique of circular p巴rmutationin

which the conventional N-terminal is connected with the

C明記ロ凶nalto generate new N-and C-terminals has been

studied.35) Therefore， the interaction of the N叫 andC-

terminals observed in this study may be applicable to

other proteins ev巴nwhen the protein does not have a (s/ α)8-barrel structure.

Th巴 authorsthank Suntory Holdings Ltd. for th巴 supplyof 'xylo-biose mixture'. We are grateful for the editorial assistance of Dr. Joanne B. Hart in the preparation of this manuscript. This work was supported in part by a Grant回in-Aidfor Scientific R巴searchof Japan Society for the Promotion of Science and the Program for Promo悶

tion of Basic Research Activities for Innovative Biosciences.

REFERENCES

1) N.C. Carpita and D.M. Gibeaut: Structural models of primary cell walls in flowering plants: consistency of molecular struc同

ture with th巴 physicalproperties of the walls during growth. Plant J.， 3， 1-30 (1993).

2) T.E. Timell: Wood hemicelluloses: part 11. in Advances in Carbohydrate Chemistry， Vol. 20， M.L. Wolfrom and R.S. Tipso， eds.， Academic Press， New York， pp. 409-483 (1965).

3) T. Collins， C. Gerday and G. Feller: Xylanas巴s，xylanase famトlies and巴xtremophilicxylanases. FEMS Microbiol. Rev.， 29， 3-23 (2005)

4) K. Ishikawa， H. Nakamura， K. Morikawa and S. Kanaya: Sta-bi1ization of Escherichia coli ribonuclease HI by cavity-fi1ling mutations within a hydrophobic core. Biochemistry， 32， 6171-6178 (1993)

5) M.K. Chan， S. Mukund， A. Kletzin， M爪T.Adams and D.C. Rees: Structure of a hyp巴rthermophilictungstopterin enzyme，

a1dehyde ferredoxin oxidoreductase. Science， 267， 1463-1469 (1995).

6) J. Sakon， W.S. Adney， M.E. Himmel， S.R. Thomas and P.A. Karplus: Crystal structure of thermostabl巴 family5 endocell日明lase El from Acidothermus cellulolyticus in complex with cel-lot巴traose.Biochemistry， 35， 10648-10660 (1996).

7) K. Ishikawa， M. Okumura， K. Katayanagi， S. Kimura， S. Kana-ya， H. Nakamura and K. Morikawa: Crystal structur巴 ofribcとnucleas巴 Hfrom刀lermusthermophilus HB8 refin巴dat 2.8 A resolution.よ Mol.Biol.， 230， 529-542 (1993).

8) J.J. Tanner， R.M. H巴chtand K.L. Krause: Det巴rminantsof en-zyme th巴rmostabilityobserved in出巴 molecularstructure of Thel7nus aquaticus D-glycerald巴hyde-3-phosphatedehydroge-nas巴 at25 angstroms resolution. Biochemistη1， 35， 2597-2609

(1996). 9) H. Nicholson， D.E. Anderson， S. Dao-pin and B.W. Matth巴ws:

Analysis of the interaction between charg巴dside chains and the alpha-helix dipole using d巴signedth巴rmostablemutants of phage T4 Iysozyme. Biochemistry， 30， 9816-9828 (1991).

10) C.M. Fontes， G.P. Hazlewood， E. Morag， J. Hall， B.H. Hirst and H.J. Gilbert: Evid巴ncefor a general role for norトcatalyticthermostabilizing domains in xylanases from thermophilic bac回

teria. Biochem. J.， 307， 151-158 (1995). 11) J.H. Clark巴， K. Davidson， H.J. Gilb巴rt，C.M. Fontes and G.P.

Hazlewood: A modular xylanas巴 frommesophilic Cellulo-monas fimi contains the same cellulose-binding and thermosta-bilizing domains as xylanases from thermophilic bacteria. FEMS Microbiol. Lett.， 139， 27-35 (1996).

12) H. Hayashi， K. Takagi， M. Fukumura， T. Kimura， S. Karita， K.

Sakka and K. Ohmiya: Sequence of xynC and prop剖 iesof XynC， a m共Jorcompon巴ntof the Clostridium thel7nocellum cellulosome.よBacteriol.， 179，4246-4253 (1997).

13) A. Blanco， P. Diaz， J. Zueco， P. Parascandola and F.I. Javier Pastor: A multidomain xylanas巴 froma Bacillus sp. with a r巴-

gion homologous to thermostabilizing domains of thermophilic enzymes. Microbiology， 145， 2163-2170 (1999).

14) S.J. Chamock， D.N. Bolam， J.P. Turk巴nburg， H.J. Gilb巴lt，L.M. Fe汀巴ira，G.J. Daωvi記巴sand C s山tab凶1註泌l訂i凶Z反in昭g"d白伽Oωma訂m郎1S0ぱfx勾yρla組naω5巴侭sare ca訂rb加oh句1ザyd合ra蹴t匂巴-七b悩in凶1吋d必曲1訂lngmodules: s託tructureand biochemistry of the Cα'lostかrバidiωuイ問mη1t仇her-mocellum X6b domain. Biochemistry， 39， 5013-5021 (2000).

15) A. Sunna， M.D. Gibbs and P.L. B巴rgquist:The th巴rmostabiliz-ing domain， XynA， of Caldibacillus celluloνorans xylanase is a xylan binding domain. Biochem. J.， 346， 583-586 (2000).

16) P.V‘Nikolova， A.L. Creagh， S.J. Duff and C.A. Haynes: Ther-mostability and irr巴V巴rsibleactivity loss of巴xoglucanase/xyla-nase Cex fr・omCellulomonas fimi. Biochemistη， 36， 1381-1388 (1997)

17) A. Ruiz-Arribas， R.I. Santamaria， G.G. Zhadan， E. Vi1lar and V.L. Shnyrov: Diff，巴rentialscanning calorim巴tricstudy of the thermal stability of xylanase from Streptomyces halstedii JM8 Biochemistry， 33， 13787-13791 (1994).

18) A. Ruiz-Arribas， G.G. Zhadan， V.P. Kutyshenko， R.I. San-tamaria， M. Co民り0，E. Villar， J.M. Fernandez-Abalos， J.J. Calvete and V.L. Shnyrov: Thermodynamic stability of two vanants

Importanc巴ofN-and C-Terminals Int巴ractionin a Xylanase 171

460，61-66 (1999). 25) S. Kan巴ko，S. Iwamatsu， A. Kuno， Z. Fujimoto， Y. Sato， K

Yura， M. Go， H. Mizuno， K. Taira， T. Hasegawa， 1. Kusakabe and K. Hayashi: Module shuffling of a family F/lO xylanas巴

replacement of modul巴sM4 and M5 of th巴 FXYNof Strepto-myces olivaceoviridis E-86 with those of the Cex of Cellulo-monas fimi. Protein Eng.， 13， 873-879 (2000).

26) A. Kuno， D. Shimizu， S. Kan巴ko，T. Hasegawa， Y. Gama， K Hayashi， 1. Kusakabe and K. Taiぽra:Si泡gn必1註ifica似如nt巴n註血har旦lcem巴n以lti加n

the binding of p-r心-ni註it佐加roph巴nyl-s凶心D-xy列lobiosid巴by th巴E128H mutant F/lO xylanas巴 fromS釘trept，ωomη1yces01μiv椛 e印ov山li仇i力ridiおsE-必86.FEBS Lett.， 450， 299-305 (1999).

27) K. Takeo， Y. Ohguchi， R. Hasegawa and S. Kitamura: Synthe-sis of 2-and 4-nitrophenyl s-glycosides of s-(l→4)-D-xylo-oligosaccharid巴sof dp 2-4. Carbohydr. Res.， 277， 231-244 (1995).

28) V. Notenboom， S.K. Williams， R. Hoos， S.G. Withers and D.R. Rose: Detail巴dstructural analysis of glycosidase/inhibitor interactions: complexes of C巴xfrom Cellulomonas fimi with xylobiose-d巴rivedaza-sugars. Biochemistly， 39， 11553-11563 (2000)

29) L. Lo Leggio， S. Kalogiannis， M.K. Bhat and R.W. Pickers但

gill: High resolution structure and sequence of T. aurantiacus xylanase 1: implications for the evolution of thermostability in family 10 xylanases and enzymes with (戸)α-barrelarchitecture. Proteins， 36， 295-306 (1999).

30) 1. Ihsanawati， T. Kumasaka， T. Kaneko， C. Morokuma， R. Ya-tsunami， T. Sato， S. Nakamura and N. Tanaka: Structural basis of the substrate subsite and the highly thermal stability of xy-lanase 10B from Thermotoga maritima MSB8. Proteins， 61， 999-1009 (2005).

31) C. Winterhalter， P. H巴inrich，A. Candussio， G. Wich and W. Li巴bl: Identification of a nov巴1cellulose-binding domain within th巴 multidomain120 kDa xylanase XynA of the hyper-thermophilic bact巴riumThermotoga maritima. Mol. Microbiol.， 15，431-444 (1995).

32) A. Ruiz-Arribas， P. Sanch巴z，J.J. Calv巴te，M. Raida， J.M Fernand巴z-Abalosand R.1. Santamana: Analysis of xysA， a gene from Streptomyces halstedii JM8 that encod巴sa 45-kilodalton modular xylanase， Xysl. Appl. Environ. Microbiol.， 63， 2983-2988 (1997).

33) H. Xie， J. Flint， M. Vardakou， J.H. Lakey， R.J. Lewis， H.J.

Gilb巴rtand C. Dumon: Probinσthe structural basis for th巴 difむ

ference in thermostability display巴dby family 10 xylanas巴s.J.

放線菌 Streptomycesolivaceoviridis E・86由来

ファミリ -10キシラナーゼの触媒ドメイン中

に存在する N末端および C末端 αヘリックスの

酵素安定性における重要性

金子哲伊藤茂泰藤本瑞久野敦2

一ノ瀬仁美岩松新之輔長谷川奥日2

l独立行政法人農業・食品産業技術総合研究機構

食品総合研究所

(305-8642つくば市観音台 2-1-12)

2山形大学理学部

(990-8560山形市小白川町 1-4-12)

3独立行政法人農業生物資源研究所

(305-8602つくば市観音台 2…1-2)

放線菌 Str・eptomycesolivaceoviridis E-86由来ファミリー

10キシラナーゼ (SoXynlOA)は触媒モジュール，糖結合

モジュールおよび両モジュールを繋ぐリンカーからなる.

われわれは，これらすべてを含む結晶構造解析に成功し

た.その結果，触媒ドメインは九つの αヘリックス (α0-

8)と八つの 9シート (sI-8)からなり，触媒ドメイン中

のN末端 αヘリックス(α0)とC末端 αヘリックス (α8)

とが相互作用していることが明らかになった.結品構造

から，両 αヘリックス聞には SoXynlOAの安定化に重要

な結合があると予想、された.そこで， SoXynlOAの安定化

に寄与している α0-α8聞の結合を明らかにするために，

SoXynlOAのC末端欠損変異体を作成し，欠損が酵素の

安定化に及ぼす影響について調べた.触媒ドメインの C

末端のアミノ駿を一つずつ削り込んだ変異体では，欠損

の程度に準じて SoXynl0Aの熱安定性が段階的に減少し

たが，酵素活性には変化がみられなかった.塩酸グアニ

ジンを用いて各変異体の安定性を比較した結果，結品構

造から予測したとおり， α8中のし巴u-300がα0と相互作

用し疎水性コアを形成していることを明らかにした.さ

らに，リンカー中の Asn-252をAlaに置換すると熱安定

性がやや減少し， α8中の Gly-303とAsn-252聞の水素結

合もまた， SoXynlOAの安定性に重要であることを明らか

にした.次に，同じファミ 1)- 10キシラナーゼである

Cellulomonas fimiの CfXynlOAとSoXynlOAとのキメラ欝

素を作成し，これらの相互作用について検証した結果， C

末端の四つのアミノ残基の付加により，熱安定性が上昇

した. しかしながら， CfXynlOAにSoXynl0AのN末端，

C末端を導入した場合には， SoXynlOAと間程度の熱安定

性の減少が確認できた.N末端と C末端の相互作用の温

度限界は SoXynlOAと一致しており，再末端のゆがみに

よりタンパク変性しやすくなることを明らかにした.