Post on 21-Jan-2022
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.
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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.